Network-based microbial compositions and methods

ABSTRACT

Provided are therapeutic compositions containing combinations of bacteria, for the maintenance or restoration of a healthy microbiota in the gastrointestinal tract of a mammalian subject, and methods for use thereof.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/777,252, filed Sep. 15, 2015, which is the National Stage ofInternational Application No. PCT/US2014/030817, filed Mar. 17, 2014,which_claims the benefit of U.S. Provisional Application No. 61/798,666,filed Mar. 15, 2013, all of which are incorporated by reference in theirentirety.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named 40681_US_sequencelisting.txt, created on May 21, 2018,with a size of 4,166,333 bytes. The sequence listing is incorporated byreference.

BACKGROUND

Mammals are colonized by microbes in the gastrointestinal (GI) tract, onthe skin, and in other epithelial and tissue niches such as the oralcavity, eye surface and vagina. The gastrointestinal tract harbors anabundant and diverse microbial community. It is a complex system,providing an environment or niche for a community of many differentspecies or organisms, including diverse strains of bacteria. Hundreds ofdifferent species can form a commensal community in the GI tract in ahealthy person, and this complement of organisms evolves from birth toultimately form a functionally mature microbial population by about 3years of age. Interactions between microbial strains in thesepopulations and between microbes and the host (e.g. the host immunesystem) shape the community structure, with availability of andcompetition for resources affecting the distribution of microbes. Suchresources may be food, location and the availability of space to grow ora physical structure to which the microbe may attach. For example, thehost's diet is involved in shaping the GI tract flora.

A healthy microbiota provides the host with multiple benefits, includingcolonization resistance to a broad spectrum of pathogens, essentialnutrient biosynthesis and absorption, and immune stimulation thatmaintains a healthy gut epithelium and an appropriately controlledsystemic immunity. In settings of dysbiosis' or disrupted symbiosis,microbiota functions can be lost or deranged, resulting in increasedsusceptibility to pathogens, altered metabolic profiles, or induction ofproinflammatory signals that can result in local or systemicinflammation or autoimmunity. Thus, the intestinal microbiota plays asignificant role in the pathogenesis of many diseases and disorders.Many of these diseases and disorders are chronic conditions thatsignificantly decrease a subject's quality of life and can be ultimatelyfatal.

Manufacturers of probiotics have asserted that their preparations ofbacteria promote mammalian health by preserving the natural microflorain the GI tract and reinforcing the normal controls on aberrant immuneresponses. See, e.g., U.S. Pat. No. 8,034,601. Probiotics, however, havebeen limited to a very narrow group of genera and a correspondinglylimited number of species. As such, they do not adequately replace themissing natural microflora nor correct dysbioses of the GI tract in manysituations.

Therefore, in response to the need for durable, efficient, and effectivecompositions and methods for prevention, diagnosis and/or treatment ofprediabetes and diabetes by way of restoring or enhancing microbiotafunctions, we address these and other shortcomings of the prior art byproviding compositions and methods for treating subjects.

SUMMARY OF THE INVENTION

Disclosed herein are methods for treating, preventing, or reducing theseverity of a disorder selected from the group consisting of Clostridiumdifficile Associated Diarrhea (CDAD), Type 2 Diabetes, Obesity,Irritable Bowel Disease (IBD), colonization with a pathogen orpathobiont, and infection with a drug-resistant pathogen or pathobiont,comprising: administering to a mammalian subject in need thereof aneffective amount of a therapeutic bacterial composition, saidtherapeutic bacterial composition comprising a plurality of isolatedbacteria or a purified bacterial preparation, the plurality of isolatedbacteria or the purified bacterial preparation capable of forming anetwork ecology selected from the group consisting of N262.S, N290.S,N284.S, N271.S, N282.S, N288.S, N302.S, N279.S, N310.S, N323.S, N331.S,N332.S, N301.S, N312.S, N339.S, N325.S, N340.S, N341.S, N346.S, N338.S,N336.S, N345.S, N355.S, N356.S, N343.S, N329.S, N361.S, N353.S, N381.S,N344.S, N352.S, N357.S, N358.S, N369.S, N372.S, N375.S, N380.S, N374.S,N377.S, N368.S, N370.S, N373.S, N376.S, N389.S, N394.S, N431.S, N434.S,N390.S, N397.S, N387.S, N440.S, N396.S, N399.S, N403.S, N414.S, N430.S,N432.S, N436.S, N437.S, N457.S, N545, N386.S, N402.S, N405.S, N415.S,N421.S, N422.S, N423.S, N458.S, N459.S, N493.S, N416.S, N439.S, N447.S,N490.S, N526, N429.S, N433.S, N448.S, N488.S, N508.S, N509.S, N510.S,N511.S, N408.S, N446.S, N451.S, N474.S, N520.S, N521.S, N535.S, N516.S,N463.S, N518.S, N586, N450.S, N465.S, N519.S, N537.S, N419.S, N468.S,N477.S, N514.S, N382.S, N460.S, N462.S, N512.S, N517.S, N523.S, N547.S,N548.S, N577.S, N581.S, N585.S, N616.S, N466.S, N469.S, N480.S, N482.S,N484.S, N515.S, N533.S, N709, N730, N478.S, N572.S, N400.S, N543.S,N582.S, N621.S, N689, N769, N481.S, N525.S, N528.S, N534.S, N574.S,N580.S, N590.S, N591.S, N597.S, N664, N693, N530.S, N687, N470.S,N529.S, N539.S, N546.S, N570.S, N579.S, N602.S, N614.S, N648.S, N652.S,N655.S, N672.S, N681.S, N690.S, N692.S, N698.S, N737.S, N738.S, N785,N841, N878, N880, N881, N987, N988, N996, N1061, N479.S, N538.S, N542.S,N578.S, N609.S, N611.S, N617.S, N666.S, N675.S, N682.S, N844, N845,N846, N852, N876, N982, N1008, N649.S, N657.S, N678.S, N686.S, N710.S,N522.S, N651.S, N653.S, N654.S, N680.S, N712.S, N792, N802, N804, N807,N849, N858, N859, N875, N885, N942, N961, N972, N1051, N587.S, N589.S,N612.S, N625.S, N656.S, N714.S, N779, N781, N828, N829, N860, N894,N925, N927, N935, N947, N983, N1023, N441.S, N584.S, N794, N788, N524.S,N604.S, N610.S, N623.S, N663.S, N669.S, N676.S, N703.S, N775.S, N777.S,N780.S, N817.S, N827.S, N836.S, N871.S, N874.S, N898.S, N907.S, N998.S,N1088, N1089, N660.S, N665.S, N667.S, N733.S, N734.S, N739.S, N741.S,N782.S, N789.S, N796.S, N798.S, N800.S, N809.S, N816.S, N842.S, N843.S,N869.S, N986.S, N995.S, N1002.S, N1004.S, N1019.S, N1093, N668.S,N685.S, N835.S, N851.S, N464.S, N695.S, N776.S, N793.S, N815.S, N833.S,N891.S, N1070.S, N1092, N795.S, N797.S, N808.S, N811.S, N826.S, N830.S,N832.S, N840.S, N945.S, N960.S, N968.S, N1091, N805.S, N822.S, N928.S,N936.S, N1078.S, and N913.S.

In some embodiments, the therapeutic bacterial composition comprises atleast one bacterial entity, wherein said bacterial entity is capable offorming the network ecology in combination with one more bacterialentities present in the gastrointestinal tract of the mammalian subjectat the time of the administering or thereafter. In certain embodiments,the network ecology is selected from the group consisting of N1008,N1023, N1051, N1061, N1070.S, N1088, N1089, N1092, N381.S, N382.S,N387.S, N399.S, N400.S, N402.S, N403.S, N414.S, N429.S, N430.S, N432.S,N433.S, N436.S, N437.S, N439.S, N441.S, N447.S, N448.S, N457.S, N460.S,N462.S, N463.S, N464.S, N470.S, N474.S, N488.S, N490.S, N493.S, N508.S,N509.S, N510.S, N511.S, N512.S, N514.S, N515.S, N517.S, N518.S, N519.S,N520.S, N523.S, N524.S, N529.S, N539.S, N543.S, N546.S, N547.S, N548.S,N570.S, N574.S, N577.S, N579.S, N580.S, N582.S, N584.S, N585.S, N589.S,N591.S, N597.S, N602.S, N604.S, N609.S, N610.S, N611.S, N612.S, N614.S,N616.S, N621.S, N623.S, N625.S, N648.S, N651.S, N652.S, N653.S, N654.S,N655.S, N660.S, N663.S, N664, N665.S, N666.S, N669.S, N672.S, N676.S,N681.S, N687, N689, N690.S, N692.S, N693, N695.S, N698.S, N703.S, N709,N712.S, N714.S, N730, N734.S, N737.S, N738.S, N769, N775.S, N777.S,N779, N780.S, N781, N785, N788, N792, N793.S, N794, N797.S, N798.S,N802, N804, N807, N817.S, N827.S, N828, N830.S, N832.S, N833.S, N836.S,N840.S, N841, N844, N845, N849, N852, N858, N859, N860, N869.S, N871.S,N874.S, N875, N878, N880, N881, N885, N894, N898.S, N907.S, N913.S,N925, N927, N942, N947, N961, N968.S, N972, N982, N983, N986.S, N987,N988, N996, and N998.S.

In one embodiment, the network ecology consists essentially of N1008,N1023, N1051, N1061, N1070.S, N1088, N1089, N1092, N381.S, N382.S,N387.S, N399.S, N400.S, N402.S, N403.S, N414.S, N429.S, N430.S, N432.S,N433.S, N436.S, N437.S, N439.S, N441.S, N447.S, N448.S, N457.S, N460.S,N462.S, N463.S, N464.S, N470.S, N474.S, N488.S, N490.S, N493.S, N508.S,N509.S, N510.S, N511.S, N512.S, N514.S, N515.S, N517.S, N518.S, N519.S,N520.S, N523.S, N524.S, N529.S, N539.S, N543.S, N546.S, N547.S, N548.S,N570.S, N574.S, N577.S, N579.S, N580.S, N582.S, N584.S, N585.S, N589.S,N591.S, N597.S, N602.S, N604.S, N609.S, N610.S, N611.S, N612.S, N614.S,N616.S, N621.S, N623.S, N625.S, N648.S, N651.S, N652.S, N653.S, N654.S,N655.S, N660.S, N663.S, N664, N665.S, N666.S, N669.S, N672.S, N676.S,N681.S, N687, N689, N690.S, N692.S, N693, N695.S, N698.S, N703.S, N709,N712.S, N714.S, N730, N734.S, N737.S, N738.S, N769, N775.S, N777.S,N779, N780.S, N781, N785, N788, N792, N793.S, N794, N797.S, N798.S,N802, N804, N807, N817.S, N827.S, N828, N830.S, N832.S, N833.S, N836.S,N840.S, N841, N844, N845, N849, N852, N858, N859, N860, N869.S, N871.S,N874.S, N875, N878, N880, N881, N885, N894, N898.S, N907.S, N913.S,N925, N927, N942, N947, N961, N968.S, N972, N982, N983, N986.S, N987,N988, N996, or N998. S.

In another embodiment, the network ecology is selected from the groupconsisting of N387.S, N399.S, N512.S, N462.S, N651.S, N982, and N845. Inone embodiment, network ecology comprises N387.S and the therapeuticbacterial composition comprises at least one bacterium selected fromeach of clade_262, clade_396, clade_444, clade_478, clade_500, andclade_553. In another embodiment, the network ecology comprises N387.Sand the therapeutic bacterial composition consists essentially of atleast one bacterium selected from each of clade_262, clade_396,clade_444, clade_478, clade_500, and clade_553. In certain embodiments,clade_262 comprises one or more bacteria selected from the groupconsisting Clostridium glycyrrhizinilyticum, Clostridium nexile,Coprococcus comes, Lachnospiraceae bacterium 1_1_57FAA, Lachnospiraceaebacterium 1_4_56FAA, Lachnospiraceae bacterium 8_1_57FAA, Ruminococcuslactaris, and Ruminococcus torques, wherein clade_396 comprises one ormore bacteria selected from the group consisting Acetivibrioethanolgignens, Anaerosporobacter mobilis, Bacteroides pectinophilus,Clostridium aminovalericum, Clostridium phytofermentans, Eubacteriumhallii, and Eubacterium xylanophilum, wherein clade_444 comprises one ormore bacteria selected from the group consisting Butyrivibriofibrisolvens, Eubacterium rectale, Eubacterium sp. oral clone GI038,Lachnobacterium bovis, Roseburia cecicola, Roseburia faecalis, Roseburiafaecis, Roseburia hominis, Roseburia intestinalis, Roseburiainulinivorans, Roseburia sp. 11SE37, Roseburia sp. 11SE38,Shuttleworthia satelles, Shuttleworthia sp. MSX8B, and Shuttleworthiasp. oral taxon G69, wherein clade_478 comprises one or more bacteriaselected from the group consisting Faecalibacterium prausnitzii,Gemmiger formicilis, and Subdoligranulum variabile, wherein clade_500comprises one or more bacteria selected from the group consistingAlistipes finegoldii, Alistipes onderdonkii, Alistipes putredinis,Alistipes shahii, Alistipes sp. HGB5, Alistipes sp. JC50, and Alistipessp. RMA 9912, and wherein clade_553 comprises one or more bacteriaselected from the group consisting Collinsella aerofaciens, Collinsellaintestinalis, Collinsella stercoris, and Collinsella tanakaei.

In one embodiment, clade_262 comprises one or more bacteria ofRuminococcus torques, wherein clade_396 comprises one or more bacteriaof Eubacterium hallii, wherein clade_444 comprises one or more bacteriaselected from the group consisting of Eubacterium rectale and Roseburiainulinivorans, wherein clade_478 comprises one or more bacteria ofFaecalibacterium prausnitzii, wherein clade_500 comprises one or morebacteria of Alistipes putredinis, and wherein clade_553 comprises one ormore bacteria of Collinsella aerofaciens.

In another embodiment, clade_262 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1048, Seq. ID No.: 1049, Seq. ID No.:1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.: 588, Seq. IDNo.: 607, and Seq. ID No.: 674, wherein clade_396 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 161, Seq. IDNo.: 288, Seq. ID No.: 551, Seq. ID No.: 6, Seq. ID No.: 613, Seq. IDNo.: 848, and Seq. ID No.: 875, wherein clade_444 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1045, Seq. IDNo.: 1634, Seq. ID No.: 1635, Seq. ID No.: 1636, Seq. ID No.: 1637, Seq.ID No.: 1638, Seq. ID No.: 1639, Seq. ID No.: 1640, Seq. ID No.: 1641,Seq. ID No.: 1728, Seq. ID No.: 1729, Seq. ID No.: 1730, Seq. ID No.:456, Seq. ID No.: 856, and Seq. ID No.: 865, wherein clade_478 comprisesone more bacteria selected from the group consisting of bacteria having16S sequences having 97% or greater identity to Seq. ID No.: 1896, Seq.ID No.: 880, and Seq. ID No.: 932, wherein clade_500 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 129, Seq. IDNo.: 131, Seq. ID No.: 132, Seq. ID No.: 133, Seq. ID No.: 134, Seq. IDNo.: 135, and Seq. ID No.: 136, and wherein clade_553 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 659, Seq. IDNo.: 660, Seq. ID No.: 661, and Seq. ID No.: 662.

In other embodiments, clade_262 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:1048, Seq. ID No.: 1049, Seq. ID No.: 1057, Seq. ID No.: 1663, Seq. IDNo.: 1670, Seq. ID No.: 588, Seq. ID No.: 607, and Seq. ID No.: 674,wherein clade_396 comprises one more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 161, Seq. IDNo.: 288, Seq. ID No.: 551, Seq. ID No.: 6, Seq. ID No.: 613, Seq. IDNo.: 848, and Seq. ID No.: 875, wherein clade_444 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences Seq. ID No.: 1045, Seq. ID No.: 1634, Seq. ID No.: 1635, Seq.ID No.: 1636, Seq. ID No.: 1637, Seq. ID No.: 1638, Seq. ID No.: 1639,Seq. ID No.: 1640, Seq. ID No.: 1641, Seq. ID No.: 1728, Seq. ID No.:1729, Seq. ID No.: 1730, Seq. ID No.: 456, Seq. ID No.: 856, and Seq. IDNo.: 865, wherein clade_478 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1896,Seq. ID No.: 880, and Seq. ID No.: 932, wherein clade_500 comprises onemore bacteria selected from the group consisting of bacteria having 16Ssequences Seq. ID No.: 129, Seq. ID No.: 131, Seq. ID No.: 132, Seq. IDNo.: 133, Seq. ID No.: 134, Seq. ID No.: 135, and Seq. ID No.: 136, andwherein clade_553 comprises one more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 659, Seq. IDNo.: 660, Seq. ID No.: 661, and Seq. ID No.: 662.

In one embodiment, clade_262 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1670, wherein clade_396 comprises oneor more bacteria selected from the group consisting of bacteria having16S sequences having 97% or greater identity to Seq. ID No.: 848,wherein clade_444 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 1639 and Seq. ID No.: 856, wherein clade_478comprises one or more bacteria selected from the group consisting ofbacteria having 16S sequences having 97% or greater identity to Seq. IDNo.: 880, wherein clade_500 comprises one or more bacteria selected fromthe group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 132, and wherein clade_553 comprisesone or more bacteria selected from the group consisting of bacteriahaving 16S sequences having 97% or greater identity to Seq. ID No.: 659.

In other embodiments, clade_262 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:1670, wherein clade_396 comprises one or more bacteria selected from thegroup consisting of bacteria having 16S sequences Seq. ID No.: 848,wherein clade_444 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 1639 and Seq.ID No.: 856, wherein clade_478 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:880, wherein clade_500 comprises one or more bacteria selected from thegroup consisting of bacteria having 16S sequences Seq. ID No.: 132, andwherein clade_553 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 659.

In another embodiment, network ecology comprises N399.S and thetherapeutic bacterial composition comprises at least one bacteriumselected from each of clade_262, clade_360, clade_396, clade_444,clade_478, and clade_494. In yet another embodiment, the network ecologycomprises N399.S and the therapeutic bacterial composition consistsessentially of at least one bacterium selected from each of clade_262,clade_360, clade_396, clade_444, clade_478, and clade_494.

In some embodiments, clade_262 comprises one or more bacteria selectedfrom the group consisting of Clostridium glycyrrhizinilyticum,Clostridium nexile, Coprococcus comes, Lachnospiraceae bacterium1_1_57FAA, Lachnospiraceae bacterium 1_4_56FAA, Lachnospiraceaebacterium 8_1_57FAA, Ruminococcus lactaris, and Ruminococcus torques,wherein clade_360 comprises one or more bacteria selected from the groupconsisting of Dorea formicigenerans, Dorea longicatena, Lachnospiraceaebacterium 2_1_46FAA, Lachnospiraceae bacterium 2_1_58FAA,Lachnospiraceae bacterium 4_1_37FAA, Lachnospiraceae bacterium9_1_43BFAA, Ruminococcus gnavus, and Ruminococcus sp. ID8, whereinclade_396 comprises one or more bacteria selected from the groupconsisting of Acetivibrio ethanolgignens, Anaerosporobacter mobilis,Bacteroides pectinophilus, Clostridium aminovalericum, Clostridiumphytofermentans, Eubacterium hallii, and Eubacterium xylanophilum,wherein clade_444 comprises one or more bacteria selected from the groupconsisting of Butyrivibrio fibrisolvens, Eubacterium rectale,Eubacterium sp. oral clone GI038, Lachnobacterium bovis, Roseburiacecicola, Roseburia faecalis, Roseburia faecis, Roseburia hominis,Roseburia intestinalis, Roseburia inulinivorans, Roseburia sp. 11SE37,Roseburia sp. 11SE38, Shuttleworthia satelles, Shuttleworthia sp. MSX8B,and Shuttleworthia sp. oral taxon G69, wherein clade_478 comprises oneor more bacteria selected from the group consisting of Faecalibacteriumprausnitzii, Gemmiger formicilis, and Subdoligranulum variabile, andwherein clade_494 comprises one or more bacteria selected from the groupconsisting of Clostridium orbiscindens, Clostridium sp. NML 04A032,Flavonifractor plautii, Pseudoflavonifractor capillosus, andRuminococcaceae bacterium D16.

In another embodiment, clade_262 comprises one or more bacteria ofRuminococcus torques, wherein clade_360 comprises one or more bacteriaof Dorea longicatena, wherein clade_396 comprises one or more bacteriaof Eubacterium hallii, wherein clade_444 comprises one or more bacteriaof Eubacterium rectale, wherein clade_478 comprises one or more bacteriaof Faecalibacterium prausnitzii, and wherein clade_494 comprises one ormore bacteria of Pseudoflavonifractor capillosus.

In one embodiment, clade_262 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1048, Seq. ID No.: 1049, Seq. ID No.:1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.: 588, Seq. IDNo.: 607, and Seq. ID No.: 674, wherein clade_360 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1050, Seq. IDNo.: 1051, Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq.ID No.: 1668, Seq. ID No.: 773, and Seq. ID No.: 774, wherein clade_396comprises one more bacteria selected from the group consisting ofbacteria having 16S sequences having 97% or greater identity to Seq. IDNo.: 161, Seq. ID No.: 288, Seq. ID No.: 551, Seq. ID No.: 6, Seq. IDNo.: 613, Seq. ID No.: 848, and Seq. ID No.: 875, wherein clade_444comprises one more bacteria selected from the group consisting ofbacteria having 16S sequences having 97% or greater identity to Seq. IDNo.: 1045, Seq. ID No.: 1634, Seq. ID No.: 1635, Seq. ID No.: 1636, Seq.ID No.: 1637, Seq. ID No.: 1638, Seq. ID No.: 1639, Seq. ID No.: 1640,Seq. ID No.: 1641, Seq. ID No.: 1728, Seq. ID No.: 1729, Seq. ID No.:1730, Seq. ID No.: 456, Seq. ID No.: 856, and Seq. ID No.: 865, whereinclade_478 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences having 97% or greater identity to Seq.ID No.: 1896, Seq. ID No.: 880, and Seq. ID No.: 932, and whereinclade_494 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences having 97% or greater identity to Seq.ID No.: 1591, Seq. ID No.: 1655, Seq. ID No.: 609, Seq. ID No.: 637, andSeq. ID No.: 886.

In some embodiments, clade_262 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1048,Seq. ID No.: 1049, Seq. ID No.: 1057, Seq. ID No.: 1663, Seq. ID No.:1670, Seq. ID No.: 588, Seq. ID No.: 607, and Seq. ID No.: 674, whereinclade_360 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1050, Seq. ID No.: 1051,Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq. ID No.:1668, Seq. ID No.: 773, and Seq. ID No.: 774, wherein clade_396comprises one more bacteria selected from the group consisting ofbacteria having 16S sequences Seq. ID No.: 161, Seq. ID No.: 288, Seq.ID No.: 551, Seq. ID No.: 6, Seq. ID No.: 613, Seq. ID No.: 848, andSeq. ID No.: 875, wherein clade_444 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:1045, Seq. ID No.: 1634, Seq. ID No.: 1635, Seq. ID No.: 1636, Seq. IDNo.: 1637, Seq. ID No.: 1638, Seq. ID No.: 1639, Seq. ID No.: 1640, Seq.ID No.: 1641, Seq. ID No.: 1728, Seq. ID No.: 1729, Seq. ID No.: 1730,Seq. ID No.: 456, Seq. ID No.: 856, and Seq. ID No.: 865, whereinclade_478 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1896, Seq. ID No.: 880,and Seq. ID No.: 932, and wherein clade_494 comprises one more bacteriaselected from the group consisting of bacteria having 16S sequences Seq.ID No.: 1591, Seq. ID No.: 1655, Seq. ID No.: 609, Seq. ID No.: 637, andSeq. ID No.: 886.

In other embodiments, clade_262 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1670, wherein clade_360 comprises oneor more bacteria selected from the group consisting of bacteria having16S sequences having 97% or greater identity to Seq. ID No.: 774,wherein clade_396 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 848, wherein clade_444 comprises one or morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 856, whereinclade_478 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 880, and wherein clade_494 comprises one ormore bacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1591.

In one aspect, clade_262 comprises one or more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1670,wherein clade_360 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 774, whereinclade_396 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 848, whereinclade_444 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 856, whereinclade_478 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 880, andwherein clade_494 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 1591.

In another aspect, the network ecology comprises N462. S and thetherapeutic bacterial composition comprises at least one bacteriumselected from each of clade_262, clade_360, and clade_478. In yetanother aspect, the network ecology comprises N462.S and the therapeuticbacterial composition consists essentially of at least one bacteriumselected from each of clade_262, clade_360, and clade_478.

In other aspects, clade_262 comprises one or more bacteria selected fromthe group consisting of Clostridium glycyrrhizinilyticum, Clostridiumnexile, Coprococcus comes, Lachnospiraceae bacterium 1_1_57FAA,Lachnospiraceae bacterium 1_4_56FAA, Lachnospiraceae bacterium8_1_57FAA, Ruminococcus lactaris, and Ruminococcus torques, whereinclade_360 comprises one or more bacteria selected from the groupconsisting of Dorea formicigenerans, Dorea longicatena, Lachnospiraceaebacterium 2_1_46FAA, Lachnospiraceae bacterium 2_1_58FAA,Lachnospiraceae bacterium 4_1_37FAA, Lachnospiraceae bacterium9_1_43BFAA, Ruminococcus gnavus, and Ruminococcus sp. ID8, and whereinclade_478 comprises one or more bacteria selected from the groupconsisting of Faecalibacterium prausnitzii, Gemmiger formicilis, andSubdoligranulum variabile.

In another aspect, clade_262 comprises one or more bacteria ofCoprococcus comes, wherein clade_360 comprises one or more bacteria ofDorea longicatena, and wherein clade_478 comprises one or more bacteriaselected from the group consisting Faecalibacterium prausnitzii andSubdoligranulum variabile.

In yet another aspect, clade_262 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1048, Seq. ID No.: 1049, Seq. ID No.:1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.: 588, Seq. IDNo.: 607, and Seq. ID No.: 674, wherein clade_360 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1050, Seq. IDNo.: 1051, Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq.ID No.: 1668, Seq. ID No.: 773, and Seq. ID No.: 774, and whereinclade_478 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences having 97% or greater identity to Seq.ID No.: 1896, Seq. ID No.: 880, and Seq. ID No.: 932.

In certain aspects, clade_262 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1048,Seq. ID No.: 1049, Seq. ID No.: 1057, Seq. ID No.: 1663, Seq. ID No.:1670, Seq. ID No.: 588, Seq. ID No.: 607, and Seq. ID No.: 674, whereinclade_360 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1050, Seq. ID No.: 1051,Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq. ID No.:1668, Seq. ID No.: 773, and Seq. ID No.: 774, and wherein clade_478comprises one more bacteria selected from the group consisting ofbacteria having 16S sequences Seq. ID No.: 1896, Seq. ID No.: 880, andSeq. ID No.: 932.

In another aspect, clade_262 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 674, wherein clade_360 comprises one ormore bacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 774, andwherein clade_478 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 1896 and Seq. ID No.: 880.

In other aspects, clade_262 comprises one or more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 674,wherein clade_360 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 774, andwherein clade_478 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 1896 and Seq.ID No.: 880.

In another embodiment, network ecology comprises N512.S and thetherapeutic bacterial composition comprises at least one bacteriumselected from each of clade_262, clade_360, and clade_444. In oneembodiment, the network ecology comprises N512.S and the therapeuticbacterial composition consists essentially of at least one bacteriumselected from each of clade_262, clade_360, and clade_444.

In other embodiments, clade_262 comprises one or more bacteria selectedfrom the group consisting of Clostridium glycyrrhizinilyticum,Clostridium nexile, Coprococcus comes, Lachnospiraceae bacterium1_1_57FAA, Lachnospiraceae bacterium 1_4_56FAA, Lachnospiraceaebacterium 8_1_57FAA, Ruminococcus lactaris, and Ruminococcus torques,wherein clade_360 comprises one or more bacteria selected from the groupconsisting of Dorea formicigenerans, Dorea longicatena, Lachnospiraceaebacterium 2_1_46FAA, Lachnospiraceae bacterium 2_1_58FAA,Lachnospiraceae bacterium 4_1_37FAA, Lachnospiraceae bacterium9_1_43BFAA, Ruminococcus gnavus, and Ruminococcus sp. ID8, and whereinclade_444 comprises one or more bacteria selected from the groupconsisting of Butyrivibrio fibrisolvens, Eubacterium rectale,Eubacterium sp. oral clone GI038, Lachnobacterium bovis, Roseburiacecicola, Roseburia faecalis, Roseburia faecis, Roseburia hominis,Roseburia intestinalis, Roseburia inulinivorans, Roseburia sp. 11SE37,Roseburia sp. 11SE38, Shuttleworthia satelles, Shuttleworthia sp. MSX8B,and Shuttleworthia sp. oral taxon G69.

In certain embodiments, clade_262 comprises one or more bacteriaselected from the group consisting of Coprococcus comes and Ruminococcustorques, wherein clade_360 comprises one or more bacteria of Dorealongicatena, and wherein clade_444 comprises one or more bacteria ofEubacterium rectale.

In one embodiment, clade_262 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1048, Seq. ID No.: 1049, Seq. ID No.:1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.: 588, Seq. IDNo.: 607, and Seq. ID No.: 674, wherein clade_360 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1050, Seq. IDNo.: 1051, Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq.ID No.: 1668, Seq. ID No.: 773, and Seq. ID No.: 774, and whereinclade_444 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences having 97% or greater identity to Seq.ID No.: 1045, Seq. ID No.: 1634, Seq. ID No.: 1635, Seq. ID No.: 1636,Seq. ID No.: 1637, Seq. ID No.: 1638, Seq. ID No.: 1639, Seq. ID No.:1640, Seq. ID No.: 1641, Seq. ID No.: 1728, Seq. ID No.: 1729, Seq. IDNo.: 1730, Seq. ID No.: 456, Seq. ID No.: 856, and Seq. ID No.: 865.

In another embodiment, clade_262 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:1048, Seq. ID No.: 1049, Seq. ID No.: 1057, Seq. ID No.: 1663, Seq. IDNo.: 1670, Seq. ID No.: 588, Seq. ID No.: 607, and Seq. ID No.: 674,wherein clade_360 comprises one more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 1050, Seq. IDNo.: 1051, Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq.ID No.: 1668, Seq. ID No.: 773, and Seq. ID No.: 774, and whereinclade_444 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1045, Seq. ID No.: 1634,Seq. ID No.: 1635, Seq. ID No.: 1636, Seq. ID No.: 1637, Seq. ID No.:1638, Seq. ID No.: 1639, Seq. ID No.: 1640, Seq. ID No.: 1641, Seq. IDNo.: 1728, Seq. ID No.: 1729, Seq. ID No.: 1730, Seq. ID No.: 456, Seq.ID No.: 856, and Seq. ID No.: 865.

In certain embodiments, clade_262 comprises one or more bacteriaselected from the group consisting of bacteria having 16S sequenceshaving 97% or greater identity to Seq. ID No.: 1670 and Seq. ID No.:674, wherein clade_360 comprises one or more bacteria selected from thegroup consisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 774, and wherein clade_444 comprises one ormore bacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 856.

In one aspect, clade_262 comprises one or more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1670and Seq. ID No.: 674, wherein clade_360 comprises one or more bacteriaselected from the group consisting of bacteria having 16S sequences Seq.ID No.: 774, and wherein clade_444 comprises one or more bacteriaselected from the group consisting of bacteria having 16S sequences Seq.ID No.: 856.

In another aspect, the network ecology comprises N845 and thetherapeutic bacterial composition comprises at least one bacteriumselected from each of clade_262, clade_360, and clade_378. In certainaspects, the network ecology comprises N845 and the therapeuticbacterial composition consists essentially of at least one bacteriumselected from each of clade_262, clade_360, and clade_378.

In other aspects, clade_262 comprises one or more bacteria selected fromthe group consisting of Clostridium glycyrrhizinilyticum, Clostridiumnexile, Coprococcus comes, Lachnospiraceae bacterium 1_1_57FAA,Lachnospiraceae bacterium 1_4_56FAA, Lachnospiraceae bacterium8_1_57FAA, Ruminococcus lactaris, and Ruminococcus torques, whereinclade_360 comprises one or more bacteria selected from the groupconsisting of Dorea formicigenerans, Dorea longicatena, Lachnospiraceaebacterium 2_1_46FAA, Lachnospiraceae bacterium 2_1_58FAA,Lachnospiraceae bacterium 4_1_37FAA, Lachnospiraceae bacterium9_1_43BFAA, Ruminococcus gnavus, and Ruminococcus sp. ID8, and whereinclade_378 comprises one or more bacteria selected from the groupconsisting of Bacteroides barnesiae, Bacteroides coprocola, Bacteroidescoprophilus, Bacteroides dorei, Bacteroides massiliensis, Bacteroidesplebeius, Bacteroides sp. 3_1_33FAA, Bacteroides sp. 3_1_40A,Bacteroides sp. 4_3_47FAA, Bacteroides sp. 9_1_42FAA, Bacteroides sp.NB_8, and Bacteroides vulgatus.

In certain aspects, clade_262 comprises one or more bacteria ofCoprococcus comes, wherein clade_360 comprises one or more bacteria ofDorea longicatena, and wherein clade_378 comprises one or more bacteriaof Bacteroides dorei.

In another aspect, clade_262 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1048, Seq. ID No.: 1049, Seq. ID No.:1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.: 588, Seq. IDNo.: 607, and Seq. ID No.: 674, wherein clade_360 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1050, Seq. IDNo.: 1051, Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq.ID No.: 1668, Seq. ID No.: 773, and Seq. ID No.: 774, and whereinclade_378 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences having 97% or greater identity to Seq.ID No.: 267, Seq. ID No.: 272, Seq. ID No.: 273, Seq. ID No.: 274, Seq.ID No.: 284, Seq. ID No.: 289, Seq. ID No.: 309, Seq. ID No.: 310, Seq.ID No.: 313, Seq. ID No.: 314, Seq. ID No.: 323, and Seq. ID No.: 331.

In certain aspects, clade_262 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1048,Seq. ID No.: 1049, Seq. ID No.: 1057, Seq. ID No.: 1663, Seq. ID No.:1670, Seq. ID No.: 588, Seq. ID No.: 607, and Seq. ID No.: 674, whereinclade_360 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1050, Seq. ID No.: 1051,Seq. ID No.: 1053, Seq. ID No.: 1058, Seq. ID No.: 1661, Seq. ID No.:1668, Seq. ID No.: 773, and Seq. ID No.: 774, and wherein clade_378comprises one more bacteria selected from the group consisting ofbacteria having 16S sequences Seq. ID No.: 267, Seq. ID No.: 272, Seq.ID No.: 273, Seq. ID No.: 274, Seq. ID No.: 284, Seq. ID No.: 289, Seq.ID No.: 309, Seq. ID No.: 310, Seq. ID No.: 313, Seq. ID No.: 314, Seq.ID No.: 323, and Seq. ID No.: 331.

In one embodiment, clade_262 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 674, wherein clade_360 comprises one ormore bacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 774, andwherein clade_378 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 274.

In another embodiment, clade_262 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:674, wherein clade_360 comprises one or more bacteria selected from thegroup consisting of bacteria having 16S sequences Seq. ID No.: 774, andwherein clade_378 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 274.

In some embodiments, the network ecology comprises N982 and thetherapeutic bacterial composition comprises at least one bacteriumselected from each of clade_172, clade_262, and clade_396. In anotherembodiment, the network ecology comprises N982 and the therapeuticbacterial composition consists essentially of at least one bacteriumselected from each of clade_172, clade_262, and clade_396.

In certain aspects, clade_172 comprises one or more bacteria selectedfrom the group consisting of Bifidobacteriaceae genomo sp. C1,Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacteriumanimalis, Bifidobacterium breve, Bifidobacterium catenulatum,Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacteriuminfantis, Bifidobacterium kashiwanohense, Bifidobacterium longum,Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum,Bifidobacterium scardovii, Bifidobacterium sp. HM2, Bifidobacterium sp.HMLN12, Bifidobacterium sp. M45, Bifidobacterium sp. MSX5B,Bifidobacterium sp. TM_7, and Bifidobacterium thermophilum, whereinclade_262 comprises one or more bacteria selected from the groupconsisting of Clostridium glycyrrhizinilyticum, Clostridium nexile,Coprococcus comes, Lachnospiraceae bacterium 1_1_57FAA, Lachnospiraceaebacterium 1_4_56FAA, Lachnospiraceae bacterium 8_1_57FAA, Ruminococcuslactaris, and Ruminococcus torques, and wherein clade_396 comprises oneor more bacteria selected from the group consisting of Acetivibrioethanolgignens, Anaerosporobacter mobilis, Bacteroides pectinophilus,Clostridium aminovalericum, Clostridium phytofermentans, Eubacteriumhallii, and Eubacterium xylanophilum.

In another aspect, clade_172 comprises one or more bacteria ofBifidobacterium longum, wherein clade_262 comprises one or more bacteriaof Coprococcus comes, and wherein clade_396 comprises one or morebacteria of Eubacterium hallii.

In one aspect, clade_172 comprises one more bacteria selected from thegroup consisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 345, Seq. ID No.: 346, Seq. ID No.: 347, Seq.ID No.: 348, Seq. ID No.: 350, Seq. ID No.: 351, Seq. ID No.: 352, Seq.ID No.: 353, Seq. ID No.: 354, Seq. ID No.: 355, Seq. ID No.: 356, Seq.ID No.: 357, Seq. ID No.: 358, Seq. ID No.: 359, Seq. ID No.: 360, Seq.ID No.: 361, Seq. ID No.: 362, Seq. ID No.: 363, Seq. ID No.: 364, andSeq. ID No.: 365, wherein clade_262 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1048, Seq. ID No.: 1049, Seq. ID No.:1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.: 588, Seq. IDNo.: 607, and Seq. ID No.: 674, and wherein clade_396 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 161, Seq. IDNo.: 288, Seq. ID No.: 551, Seq. ID No.: 6, Seq. ID No.: 613, Seq. IDNo.: 848, and Seq. ID No.: 875.

In another aspect, clade_172 comprises one more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 345,Seq. ID No.: 346, Seq. ID No.: 347, Seq. ID No.: 348, Seq. ID No.: 350,Seq. ID No.: 351, Seq. ID No.: 352, Seq. ID No.: 353, Seq. ID No.: 354,Seq. ID No.: 355, Seq. ID No.: 356, Seq. ID No.: 357, Seq. ID No.: 358,Seq. ID No.: 359, Seq. ID No.: 360, Seq. ID No.: 361, Seq. ID No.: 362,Seq. ID No.: 363, Seq. ID No.: 364, and Seq. ID No.: 365, whereinclade_262 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1048, Seq. ID No.: 1049,Seq. ID No.: 1057, Seq. ID No.: 1663, Seq. ID No.: 1670, Seq. ID No.:588, Seq. ID No.: 607, and Seq. ID No.: 674, and wherein clade_396comprises one more bacteria selected from the group consisting ofbacteria having 16S sequences Seq. ID No.: 161, Seq. ID No.: 288, Seq.ID No.: 551, Seq. ID No.: 6, Seq. ID No.: 613, Seq. ID No.: 848, andSeq. ID No.: 875.

In another aspect, clade_172 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 356, wherein clade_262 comprises one ormore bacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 674, andwherein clade_396 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 848.

In certain aspects, clade_172 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:356, wherein clade_262 comprises one or more bacteria selected from thegroup consisting of bacteria having 16S sequences Seq. ID No.: 674, andwherein clade_396 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 848.

In another embodiment, the network ecology comprises N651.S and thetherapeutic bacterial composition comprises at least one bacteriumselected from each of clade_444, clade_516, and clade_522. In yetanother embodiment, the network ecology comprises N651.S and thetherapeutic bacterial composition consists essentially of at least onebacterium selected from each of clade_444, clade_516, and clade_522.

In one embodiment, clade_444 comprises one or more bacteria selectedfrom the group consisting of Butyrivibrio fibrisolvens, Eubacteriumrectale, Eubacterium sp. oral clone GI038, Lachnobacterium bovis,Roseburia cecicola, Roseburia faecalis, Roseburia faecis, Roseburiahominis, Roseburia intestinalis, Roseburia inulinivorans, Roseburia sp.11SE37, Roseburia sp. 11SE38, Shuttleworthia satelles, Shuttleworthiasp. MSX8B, and Shuttleworthia sp. oral taxon G69, wherein clade_516comprises one or more bacteria selected from the group consisting ofAnaerotruncus colihominis, Clostridium methylpentosum, Clostridium sp.YIT 12070, Hydrogenoanaerobacterium saccharovorans, Ruminococcus albus,and Ruminococcus flavefaciens, and wherein clade_522 comprises one ormore bacteria selected from the group consisting of Bacteroidesgalacturonicus, Eubacterium eligens, Lachnospira multipara, Lachnospirapectinoschiza, and Lactobacillus rogosae. In another embodiment,clade_444 comprises one or more bacteria of Roseburia inulinivorans,wherein clade_516 comprises one or more bacteria of Anaerotruncuscolihominis, and wherein clade_522 comprises one or more bacteria ofEubacterium eligens. In some embodiments, clade_444 comprises one morebacteria selected from the group consisting of bacteria having 16Ssequences having 97% or greater identity to Seq. ID No.: 1045, Seq. IDNo.: 1634, Seq. ID No.: 1635, Seq. ID No.: 1636, Seq. ID No.: 1637, Seq.ID No.: 1638, Seq. ID No.: 1639, Seq. ID No.: 1640, Seq. ID No.: 1641,Seq. ID No.: 1728, Seq. ID No.: 1729, Seq. ID No.: 1730, Seq. ID No.:456, Seq. ID No.: 856, and Seq. ID No.: 865, wherein clade_516 comprisesone more bacteria selected from the group consisting of bacteria having16S sequences having 97% or greater identity to Seq. ID No.: 1005, Seq.ID No.: 164, Seq. ID No.: 1656, Seq. ID No.: 1660, Seq. ID No.: 606, andSeq. ID No.: 642, and wherein clade_522 comprises one more bacteriaselected from the group consisting of bacteria having 16S sequenceshaving 97% or greater identity to Seq. ID No.: 1046, Seq. ID No.: 1047,Seq. ID No.: 1114, Seq. ID No.: 280, and Seq. ID No.: 845.

In other embodiments, clade_444 comprises one more bacteria selectedfrom the group consisting of bacteria having 16S sequences Seq. ID No.:1045, Seq. ID No.: 1634, Seq. ID No.: 1635, Seq. ID No.: 1636, Seq. IDNo.: 1637, Seq. ID No.: 1638, Seq. ID No.: 1639, Seq. ID No.: 1640, Seq.ID No.: 1641, Seq. ID No.: 1728, Seq. ID No.: 1729, Seq. ID No.: 1730,Seq. ID No.: 456, Seq. ID No.: 856, and Seq. ID No.: 865, whereinclade_516 comprises one more bacteria selected from the group consistingof bacteria having 16S sequences Seq. ID No.: 1005, Seq. ID No.: 164,Seq. ID No.: 1656, Seq. ID No.: 1660, Seq. ID No.: 606, and Seq. ID No.:642, and wherein clade_522 comprises one more bacteria selected from thegroup consisting of bacteria having 16S sequences Seq. ID No.: 1046,Seq. ID No.: 1047, Seq. ID No.: 1114, Seq. ID No.: 280, and Seq. ID No.:845.

In one embodiment, clade_444 comprises one or more bacteria selectedfrom the group consisting of bacteria having 16S sequences having 97% orgreater identity to Seq. ID No.: 1639, wherein clade_516 comprises oneor more bacteria selected from the group consisting of bacteria having16S sequences having 97% or greater identity to Seq. ID No.: 164, andwherein clade_522 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences having 97% or greateridentity to Seq. ID No.: 845.

In one aspect, clade_444 comprises one or more bacteria selected fromthe group consisting of bacteria having 16S sequences Seq. ID No.: 1639,wherein clade_516 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 164, andwherein clade_522 comprises one or more bacteria selected from the groupconsisting of bacteria having 16S sequences Seq. ID No.: 845.

In another aspect, the composition further comprises apharmaceutically-acceptable excipient. In one aspect, the therapeuticbacterial composition is substantially depleted of a residual habitatproduct of a fecal material. In certain aspects, the composition isformulated for oral administration. In other embodiments, thecomposition is capable of inducing the formation of IgA, RegIII-gamma,IL-10, regulatory T cells, TGF-beta, alpha-defensin, beta-defensin, oran antimicrobial peptide in the mammalian subject. In anotherembodiment, the composition is comestible.

The invention provides a composition, comprising any of the compositionsadministered according to the methods described above. The inventionalso includes a dosage unit comprising predetermined ratios of theisolated bacteria present in the network ecology as described above.

The invention provides a method for producing short chain fatty acids(SCFA) within a mammalian subject, comprising: administering to saidmammalian subject in need thereof an effective amount of a therapeuticbacterial composition, said therapeutic bacterial composition comprisinga plurality of isolated bacteria or a purified bacterial preparation,the plurality of isolated bacteria of the purified bacterial preparationcapable of forming one or a plurality of bacterial functional pathways,the one or plurality of bacterial functional pathways capable of forminga functional network ecology selected from the group consisting ofN262.S, N290.S, N284.S, N271.S, N282.S, N288.S, N302.S, N279.S, N310.S,N323.S, N331.S, N332.S, N301.S, N312.S, N339.S, N325.S, N340.S, N341.S,N346.S, N338.S, N336.S, N345.S, N355.S, N356.S, N343.S, N329.S, N361.S,N353.S, N381.S, N344.S, N352.S, N357.S, N358.S, N369.S, N372.S, N375.S,N380.S, N374.S, N377.S, N368.S, N370.S, N373.S, N376.S, N389.S, N394.S,N431.S, N434.S, N390.S, N397.S, N387.S, N440.S, N396.S, N399.S, N403.S,N414.S, N430.S, N432.S, N436.S, N437.S, N457.S, N545, N386.S, N402.S,N405.S, N415.S, N421.S, N422.S, N423.S, N458.S, N459.S, N493.S, N416.S,N439.S, N447.S, N490.S, N526, N429.S, N433.S, N448.S, N488.S, N508.S,N509.S, N510.S, N511.S, N408.S, N446.S, N451.S, N474.S, N520.S, N521.S,N535.S, N516.S, N463.S, N518.S, N586, N450.S, N465.S, N519.S, N537.S,N419.S, N468.S, N477.S, N514.S, N382.S, N460.S, N462.S, N512.S, N517.S,N523.S, N547.S, N548.S, N577.S, N581.S, N585.S, N616.S, N466.S, N469.S,N480.S, N482.S, N484.S, N515.S, N533.S, N709, N730, N478.S, N572.S,N400.S, N543.S, N582.S, N621.S, N689, N769, N481.S, N525.S, N528.S,N534.S, N574.S, N580.S, N590.S, N591.S, N597.S, N664, N693, N530.S,N687, N470.S, N529.S, N539.S, N546.S, N570.S, N579.S, N602.S, N614.S,N648.S, N652.S, N655.S, N672.S, N681.S, N690.S, N692.S, N698.S, N737.S,N738.S, N785, N841, N878, N880, N881, N987, N988, N996, N1061, N479.S,N538.S, N542.S, N578.S, N609.S, N611.S, N617.S, N666.S, N675.S, N682.S,N844, N845, N846, N852, N876, N982, N1008, N649.S, N657.S, N678.S,N686.S, N710.S, N522.S, N651.S, N653.S, N654.S, N680.S, N712.S, N792,N802, N804, N807, N849, N858, N859, N875, N885, N942, N961, N972, N1051,N587.S, N589.S, N612.S, N625.S, N656.S, N714.S, N779, N781, N828, N829,N860, N894, N925, N927, N935, N947, N983, N1023, N441.S, N584.S, N794,N788, N524.S, N604.S, N610.S, N623.S, N663.S, N669.S, N676.S, N703.S,N775.S, N777.S, N780.S, N817.S, N827.S, N836.S, N871.S, N874.S, N898.S,N907.S, N998.S, N1088, N1089, N660.S, N665.S, N667.S, N733.S, N734.S,N739.S, N741.S, N782.S, N789.S, N796.S, N798.S, N800.S, N809.S, N816.S,N842.S, N843.S, N869.S, N986.S, N995.S, N1002.S, N1004.S, N1019.S,N1093, N668.S, N685.S, N835.S, N851.S, N464.S, N695.S, N776.S, N793.S,N815.S, N833.S, N891.S, N1070.S, N1092, N795.S, N797.S, N808.S, N811.S,N826.S, N830.S, N832.S, N840.S, N945.S, N960.S, N968.S, N1091, N805.S,N822.S, N928.S, N936.S, N1078.S, and N913.S.

In one embodiment, the functional network ecology is selected from thegroup consisting of N1008, N1023, N1051, N1061, N1070.S, N1088, N1089,N1092, N381.S, N382.S, N399.S, N400.S, N402.S, N403.S, N414.S, N429.S,N430.S, N432.S, N433.S, N436.S, N437.S, N439.S, N441.S, N447.S, N448.S,N457.S, N460.S, N462.S, N463.S, N464.S, N470.S, N474.S, N488.S, N490.S,N493.S, N508.S, N509.S, N510.S, N511.S, N512.S, N514.S, N515.S, N517.S,N518.S, N519.S, N520.S, N523.S, N524.S, N528.S, N529.S, N539.S, N543.S,N546.S, N547.S, N548.S, N570.S, N574.S, N577.S, N579.S, N580.S, N582.S,N584.S, N585.S, N589.S, N591.S, N597.S, N602.S, N604.S, N609.S, N610.S,N611.S, N612.S, N614.S, N616.S, N621.S, N623.S, N625.S, N648.S, N651.S,N652.S, N653.S, N654.S, N655.S, N660.S, N663.S, N664, N665.S, N666.S,N669.S, N672.S, N676.S, N681.S, N687, N689, N690.S, N692.S, N693,N695.S, N698.S, N703.S, N709, N712.S, N714.S, N730, N734.S, N737.S,N738.S, N769, N775.S, N777.S, N779, N780.S, N781, N785, N788, N792,N793.S, N794, N797.S, N798.S, N802, N804, N807, N817.S, N827.S, N828,N830.S, N832.S, N833.S, N836.S, N840.S, N841, N844, N845, N849, N852,N858, N859, N860, N869.S, N871.S, N874.S, N875, N878, N880, N881, N885,N894, N898.S, N907.S, N913.S, N925, N927, N942, N947, N961, N968.S,N972, N982, N983, N986.S, N987, N988, N996, and N998.S. In anotherembodiment, the functional network ecology is N528.S, and the pluralityof bacterial functional pathways comprises the functional pathways of ofKO:K00656, KO:K01069, KO:K01734, KO:K03417, KO:K03778, KO:K07246.

The invention includes a method for catalyzing secondary metabolism ofbile acids within a mammalian subject, comprising: administering to saidmammalian subject in need thereof an effective amount of a therapeuticbacterial composition, said therapeutic bacterial composition comprisinga plurality of isolated bacteria or a purified bacterial preparation,the plurality of isolated bacteria of the purified bacterial preparationcapable of forming one or a plurality of bacterial functional pathways,the one or plurality of bacterial functional pathways capable of forminga functional network ecology selected from the group consisting ofN262.S, N290.S, N284.S, N271.S, N282.S, N288.S, N302.S, N279.S, N310.S,N323.S, N331.S, N332.S, N301.S, N312.S, N339.S, N325.S, N340.S, N341.S,N346.S, N338.S, N336.S, N345.S, N355.S, N356.S, N343.S, N329.S, N361.S,N353.S, N381.S, N344.S, N352.S, N357.S, N358.S, N369.S, N372.S, N375.S,N380.S, N374.S, N377.S, N368.S, N370.S, N373.S, N376.S, N389.S, N394.S,N431.S, N434.S, N390.S, N397.S, N387.S, N440.S, N396.S, N399.S, N403.S,N414.S, N430.S, N432.S, N436.S, N437.S, N457.S, N545, N386.S, N402.S,N405.S, N415.S, N421.S, N422.S, N423.S, N458.S, N459.S, N493.S, N416.S,N439.S, N447.S, N490.S, N526, N429.S, N433.S, N448.S, N488.S, N508.S,N509.S, N510.S, N511.S, N408.S, N446.S, N451.S, N474.S, N520.S, N521.S,N535.S, N516.S, N463.S, N518.S, N586, N450.S, N465.S, N519.S, N537.S,N419.S, N468.S, N477.S, N514.S, N382.S, N460.S, N462.S, N512.S, N517.S,N523.S, N547.S, N548.S, N577.S, N581.S, N585.S, N616.S, N466.S, N469.S,N480.S, N482.S, N484.S, N515.S, N533.S, N709, N730, N478.S, N572.S,N400.S, N543.S, N582.S, N621.S, N689, N769, N481.S, N525.S, N528.S,N534.S, N574.S, N580.S, N590.S, N591.S, N597.S, N664, N693, N530.S,N687, N470.S, N529.S, N539.S, N546.S, N570.S, N579.S, N602.S, N614.S,N648.S, N652.S, N655.S, N672.S, N681.S, N690.S, N692.S, N698.S, N737.S,N738.S, N785, N841, N878, N880, N881, N987, N988, N996, N1061, N479.S,N538.S, N542.S, N578.S, N609.S, N611.S, N617.S, N666.S, N675.S, N682.S,N844, N845, N846, N852, N876, N982, N1008, N649.S, N657.S, N678.S,N686.S, N710.S, N522.S, N651.S, N653.S, N654.S, N680.S, N712.S, N792,N802, N804, N807, N849, N858, N859, N875, N885, N942, N961, N972, N1051,N587.S, N589.S, N612.S, N625.S, N656.S, N714.S, N779, N781, N828, N829,N860, N894, N925, N927, N935, N947, N983, N1023, N441.S, N584.S, N794,N788, N524.S, N604.S, N610.S, N623.S, N663.S, N669.S, N676.S, N703.S,N775.S, N777.S, N780.S, N817.S, N827.S, N836.S, N871.S, N874.S, N898.S,N907.S, N998.S, N1088, N1089, N660.S, N665.S, N667.S, N733.S, N734.S,N739.S, N741.S, N782.S, N789.S, N796.S, N798.S, N800.S, N809.S, N816.S,N842.S, N843.S, N869.S, N986.S, N995.S, N1002.S, N1004.S, N1019.S,N1093, N668.S, N685.S, N835.S, N851.S, N464.S, N695.S, N776.S, N793.S,N815.S, N833.S, N891.S, N1070.S, N1092, N795.S, N797.S, N808.S, N811.S,N826.S, N830.S, N832.S, N840.S, N945.S, N960.S, N968.S, N1091, N805.S,N822. S, N928. S, N936. S, N1078. S, and N913. S.

In one embodiment, the functional network ecology is selected from thegroup consisting of N1008, N1023, N1051, N1061, N1070.S, N1088, N1089,N1092, N381.S, N382.S, N399.S, N400.S, N402.S, N403.S, N414.S, N429.S,N430.S, N432.S, N433.S, N436.S, N437.S, N439.S, N441.S, N447.S, N448.S,N457.S, N460.S, N462.S, N463.S, N464.S, N470.S, N474.S, N488.S, N490.S,N493.S, N508.S, N509.S, N510.S, N511.S, N512.S, N514.S, N515.S, N517.S,N518.S, N519.S, N520.S, N523.S, N524.S, N529.S, N539.S, N543.S, N546.S,N547.S, N548.S, N570.S, N574.S, N577.S, N579.S, N580.S, N582.S, N584.S,N585.S, N589.S, N591.S, N597.S, N602.S, N604.S, N609.S, N610.S, N611.S,N612.S, N614.S, N616.S, N621.S, N623.S, N625.S, N648.S, N651.S, N652.S,N653.S, N654.S, N655.S, N660.S, N663.S, N664, N665.S, N666.S, N669.S,N672.S, N676.S, N681.S, N687, N689, N690.S, N692.S, N693, N695.S,N698.S, N703.S, N709, N712.S, N714.S, N730, N734.S, N737.S, N738.S,N769, N775.S, N777.S, N779, N780.S, N781, N785, N788, N792, N793.S,N794, N797.S, N798.S, N802, N804, N807, N817.S, N827.S, N828, N830.S,N832.S, N833.S, N836.S, N840.S, N841, N844, N845, N849, N852, N858,N859, N860, N869.S, N871.S, N874.S, N875, N878, N880, N881, N885, N894,N898.S, N907.S, N913.S, N925, N927, N942, N947, N961, N968.S, N972,N982, N983, N986.S, N987, N988, N996, and N998.S. In another embodiment,the functional network ecology is N660.S and the plurality of bacterialfunctional pathways comprises the functional pathways of of KO:K00656,and KO:K01442.

In some embodiment, the invention includes a composition furthercomprising a pharmaceutically-acceptable excipient. In one embodiment,the composition is formulated for oral administration. In anotherembodiment, the composition is capable of inducing the formation ofbutyrate, propionate, acetate, 7-deoxybile acids, deoxycholate acide(DCA) and lithocholic acid (LCA) in the mammalian subject. In otherembodiments, the composition is capable of inducing the depletion ofglucose, pyruvate, lactate, cellulose, fructans, starch, xylans,pectins, taurocholate, glycocholate, ursocholate, cholate,glycochenodeoxycholate, taurochenodeoxycholate, ursodeoxycholate, orchenodeoxycholate; or the formation and depletion of intermediarymetabolites acetyl-CoA, butyryl-CoA, propanoyl-CoA,chenodeoxycholoyl-CoA, or ursodeoxycholoyl-CoA in the mammalian subject.In another embodiment, the composition is formulated with one or moreprebiotic compounds. In some embodiments, the composition is comestible.

The invention includes a composition, comprising any of the compositionsadministered according to the methods described above.

The invention also includes a dosage unit comprising predeterminedratios of the isolated bacteria present in the network ecology describedabove.

The invention comprises a pharmaceutical formulation comprising apurified bacterial population consisting essentially of a bacterialnetwork capable of forming germinable bacterial spores, wherein thebacterial network is present in an amount effective to populate thegastrointestinal tract in a mammalian subject in need thereof to whomthe formulation is administered, under conditions such that at least onetype of bacteria not detectably present in the bacterial network or inthe gastrointestinal tract prior to administration is augmented.

The invention also includes a pharmaceutical formulation comprising apurified bacterial population comprising a plurality of bacterialentities, wherein the bacterial entities are present in an amounteffective to induce the formation of a functional bacterial network inthe gastrointestinal tract in a mammalian subject in need thereof towhom the formulation is administered. In some embodiments, thefunctional bacterial network comprises bacterial entities present in theformulation. In other embodiments, the functional bacterial networkcomprises bacterial entities present in the gastrointestinal tract atthe time of administration. In another embodiment, the functionalbacterial network comprises bacterial entities not present in theformulation or the gastrointestinal tract at the time of administration.In one embodiment, the formulation can be provided as an oral finishedpharmaceutical dosage form including at least one pharmaceuticallyacceptable carrier In some embodiments, the mammalian subject suffersfrom a dysbiosis comprising a gastrointestinal disease, disorder orcondition selected from the group consisting of Clostridium difficileAssociated Diarrhea (CDAD), Type 2 Diabetes, Type 1 Diabetes, Obesity,Irritable Bowel Syndrome (IBS), Irritable Bowel Disease (IBD),Ulcerative Colitis, Crohn's Disease, colitis, colonization with apathogen or pathobiont, and infection with a drug-resistant pathogen orpathobiont.

In another embodiment, the bacterial network is purified from a fecalmaterial subjected to a treatment step that comprises depleting orinactivating a pathogenic material. In one embodiment, the bacterialnetwork is substantially depleted of a detectable level of a firstpathogenic material. In some embodiments, the bacterial network issubstantially depleted of a residual habitat product of the fecalmaterial.

In one embodiment, the invention provides a method of treating orpreventing a dysbiosis in a human subject, comprising administering tothe human subject the formulation in an amount effective to treat orprevent a dysbiosis or to reduce the severity of at least one symptom ofthe dysbiosis in the human subject to whom the formulation isadministered.

In another embodiment, the formulation is provided as an oral finishedpharmaceutical dosage form including at least one pharmaceuticallyacceptable carrier, the dosage form comprising at least about 1×10⁴colony forming units of bacterial spores per dose of the composition,wherein the bacterial spores comprise at least two bacterial entitiescomprising 16S rRNA sequences at least 97% identical to the nucleic acidsequences selected from the group consisting of Seq. ID No.: 674, Seq.ID No.: 1670, Seq. ID No.: 774, Seq. ID No.: 848, Seq. ID No.: 856, Seq.ID No.: 1639, Seq. ID No.: 880, Seq. ID No.: 1896, Seq. ID No.: 1591,Seq. ID No.: 164, Seq. ID No.: 845, and Seq. ID No.: 659.

In yet another embodiment, the administration of the formulation resultsin a reduction or an elimination of at least one pathogen and/orpathobiont present in the gastrointestinal tract when the therapeuticcomposition is administered. In one embodiment, the administration ofthe formulation results in engraftment of at least one type ofspore-forming bacteria present in the therapeutic composition.

In one aspect, the administration of the formulation results inaugmentation in the gastrointestinal tract of the subject to whom theformulation is administered of at least one type of bacteria not presentin the formulation. In another aspect, the at least one type ofspore-forming bacteria are not detectably present in thegastrointestinal tract of the subject to whom the formulation isadministered when the formulation is administered. In yet anotheraspect, the administration of the formulation results in at least twoof: i) reduction or elimination of at least one pathogen and/orpathobiont present in the gastrointestinal tract when the formulation isadministered; ii) engraftment of at least one type of spore-formingbacteria present in the therapeutic composition; and iii) augmentationof at least one type of spore-forming or non-spore forming bacteria notpresent in the therapeutic composition.

In some aspects, the administration of the therapeutic compositionresults in at reduction or elimination of at least one pathogen and/orpathobiont present in the gastrointestinal tract when the therapeuticcomposition is administered and at least one of: i) engraftment of atleast one type of spore-forming bacteria present in the therapeuticcomposition; and ii) augmentation of at least one type of bacteria notpresent in the therapeutic composition.

In another aspect, the method of inducing engraftment of a bacterialpopulation in the gastrointestinal tract of a human subject, comprisingthe step of administering to the human subject an orally acceptablepharmaceutical formulation comprising a purified bacterial network,under conditions such that at least i) a subset of the spore-formingbacteria sustainably engraft within the gastrointestinal tract, or ii)at least one type of bacteria not present in the therapeutic compositionis augmented within the gastrointestinal tract.

The invention provides a pharmaceutical formulation comprising apurified first bacterial entity and a purified second bacterial entity,wherein the first bacterial entity comprises a first nucleic acidsequence encoding a first polypeptide capable of catalyzing a firstchemical reaction, wherein the second bacterial entity comprises asecond nucleic acid sequence encoding a second polypeptide capable ofcatalyzing a second chemical reaction, wherein the pharmaceuticalformulation is formulated for oral administration to a mammalian subjectin need thereof, wherein the first chemical reaction and the secondchemical reaction are capable of occurring in the gastrointestinal tractof the mammalian subject under conditions such that a first product ofthe first chemical reaction, a substance present within said mammaliansubject, or a combination of said first product with the substance isused as a substrate in the second chemical reaction to form a secondproduct, wherein the second product induces a host cell response. In oneembodiment, the substance is a mammalian subject protein or afood-derived protein. In another embodiment, the host cell responsecomprises production by the host cell of a biological material. Incertain embodiments, the biological material comprises a cytokine,growth factor or signaling polypeptide.

In one embodiment, the host cell response comprises an immune response.In another embodiment, the host cell response comprises decreasedgastric motility. In yet another embodiment, the host cell responsecomprises change in host gene expression, increased host metabolism,reduced gut permeability, enhanced epithelial cell junction integrity,reduced lipolysis by the action of Lipoprotein Lipase in adipose tissue,decreased hepatic gluconeogenesis, increased insulin sensitivity,increased production of FGF-19, or change in energy harvesting and/orstorage.

The invention includes a pharmaceutical formulation comprising apurified first bacterial entity and a purified second bacterial entity,wherein the first bacterial entity and the second bacterial entity forma functional bacterial network in the gastrointestinal tract of amammalian subject to whom the pharmaceutical formulation isadministered, wherein the functional network modulates the level and/oractivity of a biological material capable of inducing a host cellresponse.

The invention also includes a pharmaceutical formulation comprising apurified first bacterial entity and a purified second bacterial entity,wherein the first bacterial entity and the second bacterial entity forma functional bacterial network in the gastrointestinal tract of amammalian subject to whom the pharmaceutical formulation isadministered, wherein the functional network induces the production of abiological material capable of inducing a host cell response.

The invention comprises a therapeutic composition, comprising a networkof at least two bacterial entities, wherein the network comprises atleast one keystone bacterial entity and at least one non-keystonebacterial entity, wherein the at least two bacterial entities are eachprovided in amounts effective for the treatment or prevention of agastrointestinal disease, disorder or condition in a mammalian subject.In one aspect, the network comprises at least three bacterial entities.In another aspect, the network comprises at least three bacterialentities including at least two keystone bacterial entities.

The invention comprises a therapeutic composition, comprising a networkof at least two keystone bacterial entities capable of forminggermination-competent spores, wherein the at least two keystonebacterial entities are each provided in amounts effective for thetreatment or prevention of a gastrointestinal disease, disorder orcondition in a mammalian subject. In one aspect, the comprisioncomprises a network of at least two keystone bacterial entities capableof forming germination-competent spores.

In one embodiment, the invention comprises a therapeutic composition,comprising: a first network of at least two bacterial entities, whereinthe first network comprises a keystone bacterial entity and anon-keystone bacterial entity; and a second network of at least twobacterial entities, wherein the second network comprises at least onekeystone bacterial entity and at least one non-keystone bacterialentity, wherein the networks are each provided in amounts effective forthe treatment or prevention of a gastrointestinal disease, disorder orcondition in a mammalian subject.

The invention includes a therapeutic composition, comprising a networkof at least two bacterial entities, wherein the network comprises afirst keystone bacterial entity and a second keystone bacterial entity,wherein the two bacterial entities are each provided in amountseffective for the treatment or prevention of a gastrointestinal disease,disorder or condition in a mammalian subject. In one aspect, the firstand second keystone bacterial entities are present in the same network.In another aspect, the first and second keystone bacterial entities arepresent in different networks.

In some aspects, the invention comprises a diagnostic composition forthe detection of a dysbiosis, comprising a first detection moietycapable of detecting a first keystone bacterial entity and a seconddetection moiety capable of detecting a first non-keystone bacterialentity, wherein the keystone bacterial entity and the non-keystonebacterial entity comprise a network, wherein the absence of at least oneof the keystone bacterial entity and the non-keystone bacterial entityin a mammalian subject is indicative of a dysbiosis.

The invention comprises a method of altering a microbiome populationpresent in a mammalian subject, comprising the steps of determining thepresence of an incomplete network of bacterial entities in thegastrointestinal tract of the mammalian subject, and introducing to thegastrointestinal tract of the mammalian subject an effective amount ofone or more supplemental bacterial entities not detectable in thegastrointestinal tract of the mammalian subject prior to suchadministration, under conditions such that the incomplete network iscompleted, thereby altering the microbiome population.

In one embodiment, the one or more supplemental bacterial entitiesbecome part of the incomplete network, thereby forming a completenetwork. In another embodiment, the one or more supplemental bacterialentities alter the microbiota of the mammalian subject such that one ormore additional bacterial entities complete the incomplete network. Inyet another embodiment, the one or more supplemental bacterial entitiescomprise a network.

The invention includes a method for detection and correction of adysbiosis in a mammalian subject in need thereof, comprising the stepsof: providing a fecal sample from the mammalian subject comprising aplurality of bacterial entities; contacting the fecal sample with afirst detection moiety capable of detecting a first bacterial entitypresent in an network; detecting the absence of the first bacterialentity in the fecal sample, thereby detecting a dysbiosis in themammalian subject; and administering to the mammalian subject acomposition comprising an effective amount of the first bacterialentity. In one embodiment, the method includes confirming that thedysbiosis in the mammalian subject has been corrected.

The invention comprises a system for predicting a dysbiosis in asubject, the system comprising: a storage memory for storing a datasetassociated with a sample obtained from the subject, wherein the datasetcomprises content data for at least one network of bacterial entities;and a processor communicatively coupled to the storage memory fordetermining a score with an interpretation function wherein the score ispredictive of dysbiosis in the subject.

The invention also comprises a kit for diagnosis of a state of dysbiosisin a mammalian subject in need thereof, comprising a plurality ofdetection means suitable for use in detecting (1) a first bacterialentity comprising a keystone bacterial entity and (2) a second bacterialentity, wherein the first and second bacterial entities comprise afunctional network ecology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of 16S rRNA gene and denotes the coordinatesof hypervariable regions 1-9 (V1-V9), according to an embodiment of theinvention. Coordinates of V1-V9 are 69-99, 137-242, 433-497, 576-682,822-879, 986-1043, 1117-1173, 1243-1294, and 1435-1465 respectively,based on numbering using E. coli system of nomenclature defined byBrosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene(16S rRNA) from Escherichia coli, PNAS 75(10):4801-4805 (1978).

FIG. 2 highlights in bold the nucleotide sequences for eachhypervariable region in the exemplary reference E. coli 16S sequence(SEQ ID NO: 2051) described by Brosius et al.

FIG. 3 provides the OTU and clade composition of networks tested inexperiment SP-376, according to an embodiment of the invention.

FIG. 4 illustrates the results of a nutrient utilization assay withClostridium difficile and potential competitors of the pathogen. A plussign (+) indicates that it is a nutrient for the isolate tested. A minussign (−) indicates that it is not a nutrient for the isolate tested.

FIG. 5 demonstrates the microbial diversity measured in theethanol-treated spore treatment sample and patient pre- andpost-treatment samples, according to an embodiment of the invention.Total microbial diversity is defined using the Chaol Alpha-DiversityIndex and is measured at the same genomic sampling depths to confirmadequate and comparable sequence coverage of the target samples. Thepatient pretreatment (purple) harbored a microbiome that wassignificantly reduced in total diversity as compared to theethanol-treated spore treatment (red) and patient post treatment at days5 (blue), 14 (orange), and 25 (green).

FIG. 6 demonstrates how patient microbial ecology is shifted bytreatment with an ethanol-treated spore treatment from a dysbiotic stateto a state of health. Principal coordinates analysis based on the totaldiversity and structure of the microbiome (Bray Curtis Beta Diversity)of the patient pre- and post-treatment delineates that the combinationof engraftment of the OTUs from the spore treatment and the augmentationof the patient microbial ecology leads to a microbial ecology that isdistinct from both the pretreatment microbiome and the ecology of theethanol-treated spore treatment.

FIG. 7 demonstrates the augmentation of Bacteroides species in patientstreated with the spore population, according to an embodiment of theinvention.

FIG. 8 shows species engrafting versus species augmenting in patientsmicrobiomes after treatment with a bacterial composition such as but notlimited to an ethanol-treated spore population, according to anembodiment of the invention. Relative abundance of species thatengrafted or augmented as described were determined based on the numberof 16S sequence reads. Each plot is from a different patient treatedwith the bacterial composition such as but not limited to anethanol-treated spore population for recurrent C. difficile.

FIG. 9 shows a set of survival curves demonstrating efficacy of theethanol enriched spore population in a mouse prophylaxis model of C.difficile, according to an embodiment of the invention.

FIG. 10 illustrates an in vivo hamster Clostridium difficile relapseprevention model to validate efficacy of ethanol-treated spores andethanol treated, gradient purified spores, according to an embodiment ofthe invention.

FIG. 11 shows an in vivo hamster Clostridium difficile relapseprevention model to validate efficacy of network ecology bacterialcomposition, according to an embodiment of the invention.

FIG. 12 shows secondary bile acid metabolism KEGG Orthology Pathway andassociated enzymatic gene products defined by EC numbers.

FIG. 13 shows Butyrate (a.k.a butanoate) production KEGG OrthologyPathway and associated enzymatic gene products defined by EC numbers.

FIG. 14 shows Propionate (a.k.a. propanoate) production KEGG OrthologyPathway and associated enzymatic gene products defined by EC numbers.

FIG. 15 shows Acetate production KEGG Orthology Pathway and associatedenzymatic gene products defined by EC numbers.

FIG. 16 is an overview of a method to computationally derive networkecologies, according to an embodiment of the invention.

FIG. 17 is a schematic representation of how Keystone OTUs (nodes 2 and4, shaded circles) are central members of many network ecologies thatcontain non-Keystone OTUs (nodes 1, 3, and 5-9). Distinct networkecologies include [node 2--node 7], [node--3--node 2--node--4], [node2--node 4--node 5--node 6--node 7], [node 1--node 2--node 8--node 9],and [node--node 3].

FIG. 18 exemplifies a Derivation of Network Ecology Classes, accordingto an embodiment of the invention. Subsets of networks are selected foruse in defining Network Classes based on key biological criteria.Hierarchical Network clusters are defined by the presence (white) andabsence (blue) of OTUs and/or Functional Metabolic Pathways and Classesare defined as branches of the hierarchical clustering tree based on thetopological overlap measure.

FIG. 19 shows phenotypes assigned to samples for the computationalderivation of Network Ecologies that typify microbiome states of health(Hpost, Hdon, & Hgen) and states of disease (DdonF & DpreF). Thecomposition of the microbiome of samples in different phenotypes canoverlap with the intersections, defined by H, HD, D designations, havingdifferent biological meanings.

FIG. 20 shows an exemplary phylogenetic tree and the relationship ofOTUs and Clades. A, B, C, D, and E represent OTUs, also known as leavesin the tree. Clade 1 comprises OTUs A and B, Clade 2 comprises OTUs C, Dand E, and Clade 3 is a subset of Clade 2 comprising OTUs D and E. Nodesin a tree that define clades in the tree can be either statisticallysupported or not statistically supported. OTUs within a clade are moresimilar to each other than to OTUs in another clade and the robustnessthe clade assignment is denoted by the degree of statistical support fora node upstream of the OTUs in the clade.

FIG. 21 is a high-level block diagram illustrating an example of acomputer for use as a server or a user device, in accordance with oneembodiment.

The figures depict various embodiments of the present invention forpurposes of illustration only. One skilled in the art will readilyrecognize from the following discussion that alternative embodiments ofthe structures and methods illustrated herein may be employed withoutdeparting from the principles of the invention described herein.

DETAILED DESCRIPTION

Overview

Disclosed herein are therapeutic compositions containing combinations ofbacteria for the prevention, control, and treatment of gastrointestinaldiseases, and other disorders and conditions that result in or arecaused by a dysbiotic microbiome in a niche of a host. Such indicationsinclude, but are not limited to Clostridium difficile associateddiarrhea (CDAD), Type 2 Diabetes, Ulcerative colitis, as well asinfection by antibiotic resistant bacteria such as Carbapenem resistantKlebsiella pneomoniae (CRKp) and Vancomycin Resistant Enterococcus(VRE). These compositions are advantageous in being suitable for safeadministration to humans and other mammalian subjects and areefficacious in numerous gastrointestinal diseases, disorders andconditions and in general nutritional health. While bacterialcompositions are known, these are generally single bacterial strains orcombinations of bacteria that are combined without understanding theecology formed by a consortium of bacterial organisms, resulting in poorefficacy, instability, substantial variability and lack of adequatesafety.

The human body is an ecosystem in which the microbiota and themicrobiome play a significant role in the basic healthy function ofhuman systems (e.g. metabolic, immunological, and neurological). Themicrobiota and resulting microbiome comprise an ecology ofmicroorganisms that co-exist within single subjects interacting with oneanother and their host (i.e., the mammalian subject) to form a dynamicunit with inherent biodiversity and functional characteristics. Withinthese networks of interacting microbes (i.e. ecologies), particularmembers can contribute more significantly than others; as such thesemembers are also found in many different ecologies, and the loss ofthese microbes from the ecology can have a significant impact on thefunctional capabilities of the specific ecology. Robert Paine coined theconcept “Keystone Species” in 1969 (see Paine R T. 1969. A note ontrophic complexity and community stability. The American Naturalist 103:91-93.) to describe the existence of such lynchpin species that areintegral to a given ecosystem regardless of their abundance in theecological community. Paine originally describe the role of the starfishPisaster ochraceus in marine systems and since the concept has beenexperimentally validated in numerous ecosystems.

The present invention provides methods to define important networkecologies and functional network ecologies that occur in healthy anddiseased subjects, and provides the compositional constituents of thesenetwork ecologies. The method enables the derivation of ecologicalmodules (i.e. groups or networks of organisms and metabolic functions)within a broader ecology that can catalyze a change from a dysbioticmicrobiome to one that represents a state of health. In anotherembodiment the methods enable the de novo construction of a networkecology based on desired biological characteristics, includingfunctional characteristics, e.g. a functional network ecology. Themethods further provide keystone species (i.e. operational taxonomicunits, or OTUs) and keystone metabolic functions that are members ofthese microbial communities based on their ubiquitous appearance in manydifferent networks. Importantly, this method is distinguished fromprevious computational approaches by being the first method to defineactual network ecologies that are found in many healthy subjects.Network ecologies comprise consortia of bacterial OTUs (i.e. genera,species, or strains) that form coherent intact biological communitieswith defined phylogenetic and/or functional properties. In other words,the structure-function relationships contained within any NetworkEcology possess an inherent biodiversity profile and resultingbiological functional capabilities. The specific bacterial combinationsand functional capabilities of the resulting microbiome are efficaciousfor the treatment or prevention of diseases, disorders and conditions ofthe gastrointestinal tract or for the treatment or prevention ofsystemic diseases, disorders and conditions that are influenced by themicrobiota of the gastrointestinal tract. Further the network ecologieshave a modularity to their structure and function with specific nodes(as example OTUs, phylogenetic clades, functional pathways) comprising abackbone of the network onto which different r-groups (as example OTUs,phylogenetic clades, functional pathways) can be incorporated to achievespecific biological properties of the network ecology. Network Ecologiesdefined in terms of functional modalities are referred to as FunctionalNetwork Ecologies.

The network ecologies provided herein are useful in settings where amicrobial dysbiosis is occurring, given their capacities to achieve oneor more of the following actions: i) disrupting the existing microbiotaand/or microbiome; ii) establishing a new microbiota and/or microbiome;and (iii) stabilizing a functional microbiota and/or microbiome thatsupports health. Such network ecologies may be sustainably present uponintroduction into a mammalian subject, or may be transiently presentuntil such time as the functional microbiota and/or microbiome arere-established. In therapeutic settings, treatment with a consortium ofmicrobial OTUs will change the microbiome of the treated host from astate of disease to a state of health. This change in the totaldiversity and composition can be mediated by both: (i) engraftment ofOTUs that comprise the therapeutic composition into the host's ecology(Engrafted Ecology), and (ii) the establishment of OTUs that are notderived from the therapeutic composition, but for which the treatmentwith the therapeutic composition changes the environmental conditionssuch that these OTUs can establish. This Augmented Ecology is comprisedof OTUs that were present at lower levels in the host pre-treatment orthat were exogenously introduced from a source other than thetherapeutic composition itself.

Provided herein are computational methods based at least in part onnetwork theory (Proulx S R, Promislow D E L, Phillips P C. 2005. Networkthinking in ecology and evolution. Trends in Ecology & Evolution 20:345-353.), that delineate ecological and functional structures of agroup of microorganisms based on the presence or absence of the specificOTUs (i.e. microbial orders, families, genera, species or strains) orfunctions inherent to those OTUs in a population of sampled mammaliansubjects. Notably, these network ecologies and functional networkecologies are not simply inferred based on the clustering of OTUsaccording to binary co-occurrences computed from average relativeabundances across a set of subject samples (See e.g. Faust K,Sathirapongsasuti J F, Izard J, Segata N, Gevers D, Raes J, andHuttenhower C. 2012. Microbial co-occurrence relationships in the humanmicrobiome. PLoS Computational Bioliology 8: e1002606. Lozupone C, FaustK, Raes J, Faith J J, Frank D N, Zaneveld J, Gordon J I, and Knight R.2012. Identifying genomic and metabolic features that can underlie earlysuccessional and opportunistic lifestyles of human gut symbionts. GenomeResearch 22: 1974-1984), but instead the ecologies represent actualcommunities of bacterial OTUs that are computationally derived andexplicitly exist as an ecological network within one or more subjects.Further, we provide methods by which to characterize the biologicalsignificance of a given ecological network in terms of its phylogeneticdiversity, functional properties, and association with health ordisease. The present invention delineates ecologies suitable for thetreatment or prevention of diseases, disorders, and conditions of thegastrointestinal tract or which are distal to the gastrointestinal tractbut caused or perpetuated by a dysbiosis of the gut microbiota.

Definitions

As used herein, the term “purified bacterial preparation” refers to apreparation that includes bacteria that have been separated from atleast one associated substance found in a source material or anymaterial associated with the bacteria in any process used to produce thepreparation.

A “bacterial entity” includes one or more bacteria. Generally, a firstbacterial entity is distinguishable from a second bacterial entity

As used herein, the term “formation” refers to synthesis or production.

As used herein, the term “inducing” means increasing the amount oractivity of a given material as dictated by context.

As used herein, the term “depletion” refers to reduction in amount of.

As used herein, a “prebiotic” is a comestible food or beverage oringredient thereof that allows specific changes, both in the compositionand/or activity in the gastrointestinal microbiota that confers benefitsupon host well-being and health. Prebiotics may include complexcarbohydrates, amino acids, peptides, or other essential nutritionalcomponents for the survival of the bacterial composition. Prebioticsinclude, but are not limited to, amino acids, biotin,fructooligosaccharide, galactooligosaccharides, inulin, lactulose,mannan oligosaccharides, oligofructose-enriched inulin, oligofructose,oligodextrose, tagatose, trans-galactooligosaccharide, andxylooligosaccharides.

As used herein, “predetermined ratios” refer to ratios determined orselected in advance.

As used herein, “germinable bacterial spores” are spores capable offorming vegetative cells under certain environmental conditions.

As used herein, “detectably present” refers to present in an amount thatcan be detected using assays provided herein or otherwise known in theart that exist as of the filing date.

As used herein, “augmented” refers to an increase in amount and/orlocalization within to a point where it becomes detectably present.

As used herein, a “fecal material” refers to a solid waste product ofdigested food and includes feces or bowel washes.

As used herein, a “host cell response” is a response produced by a cellcomprising a host organism.

As used herein, a “mammalian subject protein” refers to a proteinproduced by a mammalian subject and encoded by the mammalian subjectgenome.

As used herein, the term “food-derived” refers to a protein found in aconsumed food.

As used herein, the term “biological material” refers to a materialproduced by a biological organism.

As used herein, the term “detection moiety” refers to an assay componentthat functions to detect an analyte.

As used herein, the term “incomplete network” refers to a partialnetwork that lacks the entire set of components needed to carry out oneor more network functions.

As used herein, the term “supplemental” refers to something that isadditional and non-identical.

As used herein, the term “Antioxidant” refers to, without limitation,any one or more of various substances such as beta-carotene (a vitamin Aprecursor), vitamin C, vitamin E, and selenium that inhibit oxidation orreactions promoted by Reactive Oxygen Species (“ROS”) and other radicaland non-radical species. Additionally, antioxidants are moleculescapable of slowing or preventing the oxidation of other molecules.Non-limiting examples of antioxidants include astaxanthin, carotenoids,coenzyme Q10 (“CoQ10”), flavonoids, glutathione, Goji (wolfberry),hesperidin, lactowolfberry, lignan, lutein, lycopene, polyphenols,selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, or combinationsthereof.

“Backbone Network Ecology” or simply “Backbone Network” or “Backbone”are compositions of microbes that form a foundational composition thatcan be built upon or subtracted from to optimize a Network Ecology orFunctional Network Ecology to have specific biological characteristicsor to comprise desired functional properties, respectively. Microbiometherapeutics can be comprised of these “Backbone Networks Ecologies” intheir entirety, or the “Backbone Networks” can be modified by theaddition or subtraction of “R-Groups” to give the network ecologydesired characteristics and properties. “R-Groups” can be defined inmultiple terms including, but not limited to: individual OTUs,individual or multiple OTUs derived from a specific phylogenetic cladeor a desired phenotype such as the ability to form spores, or functionalbacterial compositions that comprise. “Backbone Networks” can comprise acomputationally derived Network Ecology in its entirety or can besubsets of the computed network that represent key nodes in the networkthat contributed to efficacy such as but not limited to a composition ofKeystone OTUs. The number of organisms in the human gastrointestinaltract, as well as the diversity between healthy individuals, isindicative of the functional redundancy of a healthy gut microbiomeecology. See The Human Microbiome Consortia. 2012. Structure, functionand diversity of the healthy human microbiome. Nature 486: 207-214. Thisredundancy makes it highly likely that non-obvious subsets of OTUs orfunctional pathways (i.e. “Backbone Networks”) are critical tomaintaining states of health and or catalyzing a shift from a dysbioticstate to one of health. One way of exploiting this redundancy is throughthe substitution of OTUs that share a given clade (see below) or ofadding members of a clade not found in the Backbone Network.

“Bacterial Composition” refers to a consortium of microbes comprisingtwo or more OTUs. Backbone Network Ecologies, Functional NetworkEcologies, Network Classes, and Core Ecologies are all types ofbacterial compositions. A “Bacterial Composition” can also refer to acomposition of enzymes that are derived from a microbe or multiplemicrobes. As used herein, Bacterial Composition includes a therapeuticmicrobial composition, a prophylactic microbial composition, a SporePopulation, a Purified Spore Population, or ethanol treated sporepopulation.

“Clade” refers to the OTUs or members of a phylogenetic tree that aredownstream of a statistically valid node in a phylogenetic tree (FIG.20). The clade comprises a set of terminal leaves in the phylogenetictree (i.e. tips of the tree) that are a distinct monophyleticevolutionary unit and that share some extent of sequence similarity.Clades are hierarchical. In one embodiment, the node in a phylogenetictree that is selected to define a clade is dependent on the level ofresolution suitable for the underlying data used to compute the treetopology.

The “Colonization” of a host organism includes the non-transitoryresidence of a bacterium or other microscopic organism. As used herein,“reducing colonization” of a host subject's gastrointestinal tract (orany other microbiotal niche) by a pathogenic or non-pathogenic bacteriumincludes a reduction in the residence time of the bacterium thegastrointestinal tract as well as a reduction in the number (orconcentration) of the bacterium in the gastrointestinal tract or adheredto the luminal surface of the gastrointestinal tract. The reduction incolonization can be permanent or occur during a transient period oftime. Reductions of adherent pathogens can be demonstrated directly,e.g., by determining pathogenic burden in a biopsy sample, or reductionsmay be measured indirectly, e.g., by measuring the pathogenic burden inthe stool of a mammalian host.

A “Combination” of two or more bacteria includes the physicalco-existence of the two bacteria, either in the same material or productor in physically connected products, as well as the temporalco-administration or co-localization of the two bacteria.

“Cytotoxic” activity of bacterium includes the ability to kill abacterial cell, such as a pathogenic bacterial cell. A “cytostatic”activity or bacterium includes the ability to inhibit, partially orfully, growth, metabolism, and/or proliferation of a bacterial cell,such as a pathogenic bacterial cell. Cytotoxic activity may also applyto other cell types such as but not limited to Eukaryotic cells.

“Dysbiosis” refers to a state of the microbiota or microbiome of the gutor other body area, including mucosal or skin surfaces in which thenormal diversity and/or function of the ecological network is disrupted.Any disruption from the preferred (e.g., ideal) state of the microbiotacan be considered a dysbiosis, even if such dysbiosis does not result ina detectable decrease in health. This state of dysbiosis may beunhealthy, it may be unhealthy under only certain conditions, or it mayprevent a subject from becoming healthier. Dysbiosis may be due to adecrease in diversity, the overgrowth of one or more pathogens orpathobionts, symbiotic organisms able to cause disease only when certaingenetic and/or environmental conditions are present in a patient, or theshift to an ecological network that no longer provides a beneficialfunction to the host and therefore no longer promotes health.

“Ecological Niche” or simply “Niche” refers to the ecological space inwhich an organism or group of organisms occupies. Niche describes how anorganism or population or organisms responds to the distribution ofresources, physical parameters (e.g., host tissue space) and competitors(e.g., by growing when resources are abundant, and when predators,parasites and pathogens are scarce) and how it in turn alters those samefactors (e.g., limiting access to resources by other organisms, actingas a food source for predators and a consumer of prey).

“Germinant” is a material or composition or physical-chemical processcapable of inducing vegetative growth of a bacterium that is in adormant spore form, or group of bacteria in the spore form, eitherdirectly or indirectly in a host organism and/or in vitro.

“Inhibition” of a pathogen or non-pathogn encompasses the inhibition ofany desired function or activity y the bacterial compositions of thepresent invention. Demonstrations of inhibition, such as decrease in thegrowth of a pathogenic bacterium or reduction in the level ofcolonization of a pathogenic bacterium are provided herein and otherwiserecognized by one of ordinary skill in the art. Inhibition of apathogenic or non-pathogenic bacterium's “growth” may include inhibitingthe increase in size of the pathogenic or non-pathogenic bacteriumand/or inhibiting the proliferation (or multiplication) of thepathogenic or non-pathogenic bacterium. Inhibition of colonization of apathogenic or non-pathogenic bacterium may be demonstrated by measuringthe amount or burden of a pathogen before and after a treatment. An“inhibition” or the act of “inhibiting” includes the total cessation andpartial reduction of one or more activities of a pathogen, such asgrowth, proliferation, colonization, and function. Inhibition offunction includes, for example, the inhibition of expression ofpathogenic gene products such as a toxin or invasive pilus induced bythe bacterial composition.

“Isolated” encompasses a bacterium or other entity or substance that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature or in anexperimental setting), and/or (2) produced, prepared, purified, and/ormanufactured by the hand of man. Isolated bacteria include thosebacteria that are cultured, even if such cultures are not monocultures.Isolated bacteria may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they were initiallyassociated. In some embodiments, isolated bacteria are more than about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. The terms “purify,” “purifying”and “purified” refer to a bacterium or other material that has beenseparated from at least some of the components with which it wasassociated either when initially produced or generated (e.g., whether innature or in an experimental setting), or during any time after itsinitial production. A bacterium or a bacterial population may beconsidered purified if it is isolated at or after production, such asfrom a material or environment containing the bacterium or bacterialpopulation, or by passage through culture, and a purified bacterium orbacterial population may contain other materials up to about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or above about 90% and still be considered “isolated.” Insome embodiments, purified bacteria and bacterial populations are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. In the instance of bacterial compositionsprovided herein, the one or more bacterial types present in thecomposition can be independently purified from one or more otherbacteria produced and/or present in the material or environmentcontaining the bacterial type. Bacterial compositions and the bacterialcomponents thereof are generally purified from residual habitatproducts.

“Keystone OTU” or “Keystone Function” refers to one or more OTUs orFunctional Pathways (e.g. KEGG or COG pathways) that are common to manynetwork ecologies or functional network ecologies and are members ofnetworks that occur in many subjects (i.e. are pervasive). Due to theubiquitous nature of Keystone OTUs and their associated FunctionsPathways, they are central to the function of network ecologies inhealthy subjects and are often missing or at reduced levels in subjectswith disease. Keystone OTUs and their associated functions may exist inlow, moderate, or high abundance in subjects. “Non-Keystone OTU” or“non-Keystone Function” refers to an OTU or Function that is observed ina Network Ecology or a Functional Network Ecology and is not a keystoneOTU or Function.

“Microbiota” refers to the community of microorganisms that occur(sustainably or transiently) in and on an animal subject, typically amammal such as a human, including eukaryotes, archaea, bacteria, andviruses (including bacterial viruses i.e., phage).

“Microbiome” refers to the genetic content of the communities ofmicrobes that live in and on the human body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)), wherein “genetic content”includes genomic DNA, RNA such as ribosomal RNA, the epigenome,plasmids, and all other types of genetic information.

“Microbial Carriage” or simply “Carriage” refers to the population ofmicrobes inhabiting a niche within or on humans. Carriage is oftendefined in terms of relative abundance. For example, OTU1 comprises 60%of the total microbial carriage, meaning that OTU1 has a relativeabundance of 60% compared to the other OTUs in the sample from which themeasurement was made. Carriage is most often based on genomic sequencingdata where the relative abundance or carriage of a single OTU or groupof OTUs is defined by the number of sequencing reads that are assignedto that OTU/s relative to the total number of sequencing reads for thesample. Alternatively, Carriage may be measured using microbiologicalassays.

“Microbial Augmentation” or simply “augmentation” refers to theestablishment or significant increase of a population of microbes thatare (i) absent or undetectable (as determined by the use of standardgenomic and microbiological techniques) from the administeredtherapeutic microbial composition, (ii) absent, undetectable, or presentat low frequencies in the host niche (for example: gastrointestinaltract, skin, anterior-nares, or vagina) before the delivery of themicrobial composition, and (iii) are found after the administration ofthe microbial composition or significantly increased, for instance2-fold, 5-fold, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, or greaterthan 1×10⁸, in cases where they were present at low frequencies. Themicrobes that comprise an augmented ecology can be derived fromexogenous sources such as food and the environment, or grow out frommicro-niches within the host where they reside at low frequency. Theadministration of a bacterial microbial composition induces anenvironmental shift in the target niche that promotes favorableconditions for the growth of these commensal microbes. In the absence oftreatment with a bacterial composition, the host can be constantlyexposed to these microbes; however, sustained growth and the positivehealth effects associated with the stable population of increased levelsof the microbes comprising the augmented ecology are not observed.

“Microbial Engraftment” or simply “engraftment” refers to theestablishment of OTUs present in the bacterial composition in a targetniche that are absent in the treated host prior to treatment. Themicrobes that comprise the engrafted ecology are found in thetherapeutic microbial composition and establish as constituents of thehost microbial ecology upon treatment. Engrafted OTUs can establish fora transient period of time, or demonstrate long-term stability in themicrobial ecology that populates the host post treatment with abacterial composition. The engrafted ecology can induce an environmentalshift in the target niche that promotes favorable conditions for thegrowth of commensal microbes capable of catalyzing a shift from adysbiotic ecology to one representative of a health state.

As used herein, the term “Minerals” is understood to include boron,calcium, chromium, copper, iodine, iron, magnesium, manganese,molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin,vanadium, zinc, or combinations thereof.

“Network Ecology” refers to a consortium of clades or OTUs that co-occurin some number of subjects. As used herein, a “network” is definedmathematically by a graph delineating how specific nodes (i.e. clades orOTUs) and edges (connections between specific clades or OTUs) relate toone another to define the structural ecology of a consortium of cladesor OTUs. Any given Network Ecology will possess inherent phylogeneticdiversity and functional properties. A Network Ecology can also bedefined in terms of its functional capabilities where for example thenodes would be comprised of elements such as, but not limited to,enzymes, clusters of orthologous groups (COGS;ncbi.nlm.nih.gov/books/NBK21090/), or KEGG Orthology Pathways(genome.jp/kegg/); these networks are referred to as a “FunctionalNetwork Ecology”. Functional Network Ecologies can be reduced topractice by defining the group of OTUs that together comprise thefunctions defined by the Functional Network Ecology.

“Network Class” and “Network Class Ecology” refer to a group of networkecologies that in general are computationally determined to compriseecologies with similar phylogenetic and/or functional characteristics. ANetwork Class therefore contains important biological features, definedeither phylogenetically or functionally, of a group (i.e., a cluster) ofrelated network ecologies. One representation of a Network Class Ecologyis a designed consortium of microbes, typically non-pathogenic bacteria,that represents core features of a set of phylogenetically orfunctionally related network ecologies seen in many different subjects.In many occurrences, a Network Class, while designed as describedherein, exists as a Network Ecology observed in one or more subjects.Network Class ecologies are useful for reversing or reducing a dysbiosisin subjects where the underlying, related Network Ecology has beendisrupted. Exemplary Network Classes are provided in Table 12 andexamples of phylogenetic signature by family of Network Classes aregiven in Table 13.

To be free of “non-comestible products” means that a bacterialcomposition or other material provided herein does not have asubstantial amount of a non-comestible product, e.g., a product ormaterial that is inedible, harmful or otherwise undesired in a productsuitable for administration, e.g., oral administration, to a humansubject. Non-comestible products are often found in preparations ofbacteria from the prior art.

“Operational taxonomic units” and “OTU” (or plural, “OTUs”) refer to aterminal leaf in a phylogenetic tree and is defined by a nucleic acidsequence, e.g., the entire genome, or a specific genetic sequence, andall sequences that share sequence identity to this nucleic acid sequenceat the level of species. In some embodiments the specific geneticsequence may be the 16S sequence or a portion of the 16S sequence. Inother embodiments, the entire genomes of two entities are sequenced andcompared. In another embodiment, select regions such as multilocussequence tags (MLST), specific genes, or sets of genes may begenetically compared. In 16S embodiments, OTUs that share ≥97% averagenucleotide identity across the entire 16S or some variable region of the16S are considered the same OTU. See e.g. Claesson M J, Wang Q,O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P, and O'Toole P W. 2010.Comparison of two next-generation sequencing technologies for resolvinghighly complex microbiota composition using tandem variable 16S rRNAgene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A,and Tiedje J M. 2006. The bacterial species definition in the genomicera. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. In embodimentsinvolving the complete genome, MLSTs, specific genes, other than 16S, orsets of genes OTUs that share ≥95% average nucleotide identity areconsidered the same OTU. See e.g., Achtman M, and Wagner M. 2008.Microbial diversity and the genetic nature of microbial species. Nat.Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. PhilosTrans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently definedby comparing sequences between organisms. Generally, sequences with lessthan 95% sequence identity are not considered to form part of the sameOTU. OTUs may also be characterized by any combination of nucleotidemarkers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Such characterizationemploys, e.g., WGS data or a whole genome sequence.

Table 1 below shows a List of Operational Taxonomic Units (OTU) withtaxonomic assignments made to Genus, Species, and Phylogenetic Clade.Clade membership of bacterial OTUs is based on 16S sequence data. Cladesare defined based on the topology of a phylogenetic tree that isconstructed from full-length 16S sequences using maximum likelihoodmethods familiar to individuals with ordinary skill in the art ofphylogenetics. Clades are constructed to ensure that all OTUs in a givenclade are: (i) within a specified number of bootstrap supported nodesfrom one another, and (ii) within 5% genetic similarity. OTUs that arewithin the same clade can be distinguished as genetically andphylogenetically distinct from OTUs in a different clade based on 16S-V4sequence data, while OTUs falling within the same clade are closelyrelated. OTUs falling within the same clade are evolutionarily closelyrelated and may or may not be distinguishable from one another using16S-V4 sequence data. Members of the same clade, due to theirevolutionary relatedness, play similar functional roles in a microbialecology such as that found in the human gut. Compositions substitutingone species with another from the same clade are likely to haveconserved ecological function and therefore are useful in the presentinvention. All OTUs are denoted as to their putative capacity to formspores and whether they are a Pathogen or Pathobiont (see Definitionsfor description of “Pathobiont”). NIAID Priority Pathogens are denotedas ‘Category-A’, ‘Category-B’, or ‘Category-C’, and OpportunisticPathogens are denoted as ‘OP’. OTUs that are not pathogenic or for whichtheir ability to exist as a pathogen is unknown are denoted as ‘N’. The‘SEQ ID Number’ denotes the identifier of the OTU in the SequenceListing File and ‘Public DB Accession’ denotes the identifier of the OTUin a public sequence repository.

“Pathobiont” refer to specific bacterial species found in healthy hoststhat may trigger immune-mediated pathology and/or disease in response tocertain genetic or environmental factors (Chow et al. 2011. Curr OpImmunol. Pathobionts of the intestinal microbiota and inflammatorydisease. 23: 473-80). Thus, a pathobiont is an opportunistic microbethat is mechanistically distinct from an acquired infectious organism.Thus, the term “pathogen” includes both acquired infectious organismsand pathobionts.

“Pathogen”, “pathobiont” and “pathogenic” in reference to a bacterium orany other organism or entity that includes any such organism or entitythat is capable of causing or affecting a disease, disorder or conditionof a host organism containing the organism or entity, including but notlimited to pre-diabetes, type 1 diabetes or type 2 diabetes.

“Phenotype” refers to a set of observable characteristics of anindividual entity. As example an individual subject may have a phenotypeof “health” or “disease”. Phenotypes describe the state of an entity andall entities within a phenotype share the same set of characteristicsthat describe the phenotype. The phenotype of an individual results inpart, or in whole, from the interaction of the entity's genome and/ormicrobiome with the environment, especially including diet.

“Phylogenetic Diversity” is a biological characteristic that refers tothe biodiversity present in a given Network Ecology or Network ClassEcology based on the OTUs that comprise the network. Phylogeneticdiversity is a relative term, meaning that a Network Ecology or NetworkClass that is comparatively more phylogenetically diverse than anothernetwork contains a greater number of unique species, genera, andtaxonomic families. Uniqueness of a species, genera, or taxonomic familyis generally defined using a phylogenetic tree that represents thegenetic diversity all species, genera, or taxonomic families relative toone another. In another embodiment phylogenetic diversity may bemeasured using the total branch length or average branch length of aphylogenetic tree. Phylogenetic Diversity may be optimized in abacterial composition by including a wide range of biodiversity.

“Phylogenetic tree” refers to a graphical representation of theevolutionary relationships of one genetic sequence to another that isgenerated using a defined set of phylogenetic reconstruction algorithms(e.g. parsimony, maximum likelihood, or Bayesian). Nodes in the treerepresent distinct ancestral sequences and the confidence of any node isprovided by a bootstrap or Bayesian posterior probability, whichmeasures branch uncertainty.

“Prediabetes” refers a condition in which blood glucose levels arehigher than normal, but not high enough to be classified as diabetes.Individuals with pre-diabetes are at increased risk of developing type 2diabetes within a decade. According to CDC, prediabetes can be diagnosedby fasting glucose levels between 100-125 mg/dL, 2 hour post-glucoseload plasma glucose in oral glucose tolerance test (OGTT) between 140and 199 mg/dL, or hemoglobin A1c test between 5.7%-6.4%.

“rDNA”, “rRNA”, “16S-rDNA”, “16S-rRNA”, “16S”, “16S sequencing”,“16S-NGS”, “18S”, “18S-rRNA”, “18S-rDNA”, “18S sequencing”, and“18S-NGS” refer to the nucleic acids that encode for the RNA subunits ofthe ribosome. rDNA refers to the gene that encodes the rRNA thatcomprises the RNA subunits. There are two RNA subunits in the ribosometermed the small subunit (SSU) and large subunit (LSU); the RNA geneticsequences (rRNA) of these subunits is related to the gene that encodesthem (rDNA) by the genetic code. rDNA genes and their complementary RNAsequences are widely used for determination of the evolutionaryrelationships amount organisms as they are variable, yet sufficientlyconserved to allow cross organism molecular comparisons. Typically 16SrDNA sequence (approximately 1542 nucleotides in length) of the 30S SSUis used for molecular-based taxonomic assignments of Prokaryotes and the18S rDNA sequence (approximately 1869 nucleotides in length) of 40S SSUis used for Eukaryotes. 16S sequences are used for phylogeneticreconstruction as they are in general highly conserved, but containspecific hypervariable regions that harbor sufficient nucleotidediversity to differentiate genera and species of most bacteria.

“Residual habitat products” refers to material derived from the habitatfor microbiota within or on a human or animal. For example, microbiotalive in feces in the gastrointestinal tract, on the skin itself, insaliva, mucus of the respiratory tract, or secretions of thegenitourinary tract (i.e., biological matter associated with themicrobial community). Substantially free of residual habitat productsmeans that the bacterial composition no longer contains the biologicalmatter associated with the microbial environment on or in the human oranimal subject and is 100% free, 99% free, 98% free, 97% free, 96% free,or 95% free of any contaminating biological matter associated with themicrobial community. Residual habitat products can include abioticmaterials (including undigested food) or it can include unwantedmicroorganisms. Substantially free of residual habitat products may alsomean that the bacterial composition contains no detectable cells from ahuman or animal and that only microbial cells are detectable. In oneembodiment, substantially free of residual habitat products may alsomean that the bacterial composition contains no detectable viral(including bacterial viruses (i.e., phage)), fungal, mycoplasmalcontaminants. In another embodiment, it means that fewer than 1×10-2%,1×10-3%, 1×10-4%, 1×10-5%, 1×10-6%, 1×10-7%, 1×10-8 of the viable cellsin the bacterial composition are human or animal, as compared tomicrobial cells. There are multiple ways to accomplish this degree ofpurity, none of which are limiting. Thus, contamination may be reducedby isolating desired constituents through multiple steps of streaking tosingle colonies on solid media until replicate (such as, but not limitedto, two) streaks from serial single colonies have shown only a singlecolony morphology. Alternatively, reduction of contamination can beaccomplished by multiple rounds of serial dilutions to single desiredcells (e.g., a dilution of 10-8 or 10-9), such as through multiple10-fold serial dilutions. This can further be confirmed by showing thatmultiple isolated colonies have similar cell shapes and Gram stainingbehavior. Other methods for confirming adequate purity include geneticanalysis (e.g. PCR, DNA sequencing), serology and antigen analysis,enzymatic and metabolic analysis, and methods using instrumentation suchas flow cytometry with reagents that distinguish desired constituentsfrom contaminants.

“Spore” or a population of “spores” includes bacteria (or othersingle-celled organisms) that are generally viable, more resistant toenvironmental influences such as heat and bacteriocidal agents thanvegetative forms of the same bacteria, and typically capable ofgermination and out-growth. Spores are characterized by the absence ofactive metabolism until they respond to specific environmental signals,causing them to germinate. “Spore-formers” or bacteria “capable offorming spores” are those bacteria containing the genes and othernecessary abilities to produce spores under suitable environmentalconditions. A Table of preferred spore-forming bacterial compositions isprovided in Table 11.

“Spore population” refers to a plurality of spores present in acomposition. Synonymous terms used herein include spore composition,spore preparation, ethanol-treated spore fraction and spore ecology. Aspore population may be purified from a fecal donation, e.g. via ethanolor heat treatment, or a density gradient separation or any combinationof methods described herein to increase the purity, potency and/orconcentration of spores in a sample. Alternatively, a spore populationmay be derived through culture methods starting from isolated sporeformer species or spore former OTUs or from a mixture of such species,either in vegetative or spore form.

In one embodiment, the spore preparation comprises spore forming specieswherein residual non-spore forming species have been inactivated bychemical or physical treatments including ethanol, detergent, heat,sonication, and the like; or wherein the non-spore forming species havebeen removed from the spore preparation by various separations stepsincluding density gradients, centrifugation, filtration and/orchromatography; or wherein inactivation and separation methods arecombined to make the spore preparation. In yet another embodiment, thespore preparation comprises spore forming species that are enriched overviable non-spore formers or vegetative forms of spore formers. In thisembodiment, spores are enriched by 2-fold, 5-fold, 10-fold, 50-fold,100-fold, 1000-fold, 10,000-fold or greater than 10,000-fold compared toall vegetative forms of bacteria. In yet another embodiment, the sporesin the spore preparation undergo partial germination during processingand formulation such that the final composition comprises spores andvegetative bacteria derived from spore forming species.

“Sporulation induction agent” is a material or physical-chemical processthat is capable of inducing sporulation in a bacterium, either directlyor indirectly, in a host organism and/or in vitro.

To “increase production of bacterial spores” includes an activity or asporulation induction agent. “Production” includes conversion ofvegetative bacterial cells into spores and augmentation of the rate ofsuch conversion, as well as decreasing the germination of bacteria inspore form, decreasing the rate of spore decay in vivo, or ex vivo, orto increasing the total output of spores (e.g. via an increase involumetric output of fecal material).

“Subject” refers to any animal subject including humans, laboratoryanimals (e.g., primates, rats, mice), livestock (e.g., cows, sheep,goats, pigs, turkeys, and chickens), and household pets (e.g., dogs,cats, and rodents). The subject may be suffering from a dysbiosis, thatcontributes to or causes a condition classified as diabetes orpre-diabetes, including but not limited to mechanisms such as metabolicendotoxemia, altered metabolism of primary bile acids, immune systemactivation, or an imbalance or reduced production of short chain fattyacids including butyrate, propionate, acetate, and branched chain fattyacids.

As used herein the term “Vitamin” is understood to include any ofvarious fat-soluble or water-soluble organic substances (non-limitingexamples include Vitamin A, Vitamin B1 (thiamine), Vitamin B2(riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5(pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine,or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folicacid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin invitamin supplements), Vitamin C, Vitamin D, Vitamin E, Vitamin K, K1 andK2 (i.e., MK-4, MK-7), folic acid and biotin) essential in minuteamounts for normal growth and activity of the body and obtainednaturally from plant and animal foods or synthetically made,pro-vitamins, derivatives, analogs.

“V1-V9 regions” or “16S V1-V9 regions” refers to the 16S rRNA refers tothe first through ninth hypervariable regions of the 16S rRNA gene thatare used for genetic typing of bacterial samples. These regions inbacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682,822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. Brosius et al.,Complete nucleotide sequence of a 16S ribosomal RNA gene fromEscherichia coli, PNAS 75(10):4801-4805 (1978). In some embodiments, atleast one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions are usedto characterize an OTU. In one embodiment, the V1, V2, and V3 regionsare used to characterize an OTU. In another embodiment, the V3, V4, andV5 regions are used to characterize an OTU. In another embodiment, theV4 region is used to characterize an OTU. A person of ordinary skill inthe art can identify the specific hypervariable regions of a candidate16S rRNA by comparing the candidate sequence in question to a referencesequence and identifying the hypervariable regions based on similarityto the reference hypervariable regions, or alternatively, one can employWhole Genome Shotgun (WGS) sequence characterization of microbes or amicrobial community.

Interactions Between Microbiome and Host

Interactions between the human microbiome and the host shape host healthand disease via multiple mechanisms, including the provision ofessential functions by the microbiota. Examples of these mechanismsinclude but are not limited to the function of the microbiota inensuring a healthy level of bile acid metabolism, energy harvesting andstorage, and regulation of immune responses, and reducing deleteriousand unhealthy levels of gut permeability or metabolic endotoxemia.

Importance of Bile Acids to Human Health and Role of Microbiota

Primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA)are synthesized from cholesterol in the liver in humans. The synthesizedprimary bile acids are conjugated to glycine, taurine, or sulfate beforesecretion into the bile by specific transporters located in thebasolateral membrane of the hepatocyte. The ingestion of a meal triggersthe release of bile from the gallbladder into the intestinal lumen,where bile acids form micelles with dietary lipids and lipid-solublevitamins, facilitating their absorption. ˜95% of bile acids arereabsorbed via specific transporters expressed in the distal ileum, andthe remaining 5% escapes the enterohepatic cycle and travels towards thelarge intestine to be excreted in the feces. In the colon, the bileacids may undergo deconjugation and dehydroxylation by the gutmicroflora. The resulting secondary bile acids are mainly deoxycholicacid (DCA) and lithocholic acid (LCA). The bile acid pool undergoes thisenterohepatic cycle around 12×/day in humans. Although the bile acidpool size is constant, the flux of bile acids varies during the day;bile acid flux and plasma bile acid concentrations are highestpostprandially (See reviews Prawitt, J et al. 2011 Curr Diab Rep Bileacid metabolism and the pathogenesis of type 2 diabetes 11: 160-166;Nieuwdorp et al. 2014 Gastroenterology. Role of the Microbiome in EnergyRegulation and Metabolism. pii: S0016-5085(14)00219-4. doi:10.1053/j.gastro.2014.02.008).

Commensal bacteria are involved in processing primary bile acids tosecondary bile acids in the colon. Known biotransformations of bileacids by commensal GI bacteria include deconjugation of the conjugatedbile salts to liberate free bile acids and amino acid moieties, removalof hydroxyl groups principally the C-7 hydroxyl group of the cholic acidmoiety, oxidative and reductive reactions of the existing hydroxylgroups, and epimerization of bile acids.

The canonical first step in bile acid metabolism is deconjugation of thetaurine or glycine group through enzymes termed bile salt hydrolases, toyield CA and CDCA. These bile acids are then substrates for a series ofenzymatic steps that remove the 7-alphahydroxy group to form deoxycholicacid (DCA) and lithocholic acid (LCA). LCA has particularly lowsolubility due to the loss of hydrophilic side chains compared to any ofthe other bile acids. It is also feasible for microbes to dehydroxylatethe conjugated primary bile acids, giving rise to gluco-DCA; gluco-LCA;tauro-DCA; and tauro-LCA. Further modifications are possible, includingthe microbial conversion of CDCA to a 7-betahydroxy epimer,ursodeoxycholic acid (UDCA). Many other secondary bile acids are made insmaller amounts by the gut microbiota, for example, alpha-, beta-,gamma-, and omega-muricholic acids and many others (see Swann J R etal., 2011 PNAS Systemic gut microbial modulation of bile acid metabolismin host tissue compartments 108: 4523-30).

Intestinal microbiota play a key role in bile acid metabolism. Germ-freemice have altered metabolism of bile acids, including increased levelsof conjugated bile acids throughout the intestine, with nodeconjugation, and strongly decreased fecal excretion. Provision ofampicillin to mice increases biliary bile acid output 3-fold anddecreases fecal output by 70%.

Dysbiosis of the gut microbiome affecting bile acid metabolism mayaffect adiposity, glucose regulation, and inflammation, among othereffects. Bile acids are essential solubilizers of lipids, fats, andlipid soluble vitamins to enhance absorption of nutrients in the smallintestine, and are also signaling molecules that regulate metabolism,including glucose homeostasis and basal metabolic rate. See Houten, S Met al. 2006 EMBO J. Endocrine function of bile acids. 25: 1419-25;Prawitt, J et al. 2011 Curr Diab Rep. Bile acid metabolism and thepathogenesis of type 2 diabetes. 11: 160-166. For example, bile acidsequestrants (non-absorbable polymers that complex bile acids in theintestinal lumen and divert them from the enterohepatic cycle) canimprove glycemic control in Type 2 diabetes patients. Prawitt, J et al.2011 Curr Diab Rep Bile acid metabolism and the pathogenesis of type 2diabetes 11: 160-166.

The most prominent targets of action by bile acids and their metabolitesinclude FXR (farnesoid X receptor), an orphan nuclear receptor withinthe liver and intestine, and TGR5, a G-protein coupled receptor found ongallbladder, ileum, colon, brown and white adipose tissue. FXR ispreferentially activated by CDCA, and in turn upregulates the expressionof gene products including FGF-19 (fibroblast growth factor 19) inhumans. FGF-15 (the murine analogue of FGF-19) increases basal metabolicrate and reverses weight gain in mice given a high fat diet. FXRactivation also down-regulates hepatic gluconeogenesis. Although bothconjugated and unconjugated bile acids can bind to FXR, the conjugatedforms must be actively transported into the cell to initiate signalingwhereas the unconjugated bile acids can diffuse through the membraneowing to their lower molecular weight, higher pKa and tendency to existin the protonated form.

TGR5 is preferentially activated by the secondary bile acid LCA andtauro-LCA with downstream effects, among others, on expression ofincretin hormones such as GLP-1 that modulate insulin production andhelp maintain glucose homeostasis.

Bile acids are therefore important metabolic regulators. Additionalinsight into the importance of the interplay between the gut microbiome,bile acid metabolism, and glucose homeostasis is provided by theobservation that treating obese male patients with a 7-day course ofvancomycin decreases total microbiota diversity, specifically depletingspecies in the diverse Clostridium IV and XIVa clusters. Among theClostridia are various organisms that metabolize bile acids as well asothers that produce short chain fatty acids, including butyrate andpropionate. In contrast, treatment with a 7-day course of amoxicillinproduces a trend toward increased microbiota diversity. Moreover, fecalbile acid composition is markedly changed following vancomycintreatment; secondary bile acids DCA, LCA and iso-LCA decrease whereasprimary bile acids CA and CDCA increase. Amoxicillin treatment does notalter the ratio of bile acids in fecal samples. FGF-19 levels in serumare also decreased following vancomycin treatment, but not amoxicillintreatment. Peripheral insulin sensitivity changes following vancomycinbut not amoxicillin treatment. Vrieze, A et al., 2013 J Hepatol Impactof oral vancomycin on gut microbiota, bile acid metabolism, and insulinsensistivity dx.doi.org/10.1016/j.jhep.2013.11.034.

While the study by Vrieze et al. points out the potential for microbesto improve insulin homeostasis through enhanced secondary bile acidmetabolism, the authors point out several limitations of their work.Most importantly, while the HIT-Chip analysis used to generate fecalmicrobial profiles provides valuable information regarding classes oforganisms, it does not provide mechanistic information or identifyspecific species or functional enzymatic pathways responsible for theobserved effects. Moreover, the HIT-Chip is a hybridization based assayand the similarity of sequences among the organisms in the Clostridialclusters may lead to mis-assignments. As a result, others have failed todefine specific compositions that can be used to modulate insulinsensitivity via bile acid metabolism.

In addition to the role for bile acids as metabolic regulators, bileacids are also linked to inflammatory disease. Crohn's Disease patientsin the Metagenomics of the Human Intestinal Tract (MetaHIT) cohort showreduced bile salt hydrolase (BSH) gene abundance compared to patientswithout disease, and increased primary bile acids in inflammatory bowelsyndrome patients is correlated with stool frequency and consistency(Duboc et al. 2012 Neurogastroenterol Motil. Increase in fecal primarybile acids and dysbiosis in patients with diarrhea-predominant irritablebowel syndrome. doi: 10.1111/j.1365-2982.2012.01893.x). Furthermore,TGR5 is expressed on immune monocytes and macrophages in addition to GIand liver tissues, and FXR and TGR5 are known to be involved inregulation of inflammation in enterohepatic tissues (Jones et al., 2014Expert Opin Biol Ther The human microbiome and bile acid metabolism:dysbiosis, dysmetabolism, disease and interventiondoi:10.1517/14712598.2014.880420)

Multiple methods are available for determination of bile acids in serum,bile and faces of individuals. As reviewed by Sharma (Sharma, K R,Review on bile acid analysis, Int J Pharm Biomed Sci 2012, 3(2), 28-34),a variety of methods can be used, such as chemical (Carey J B, et al,1958, The bile acids in normal human serum with comparative observationsin patients with jaundice. J Lab Clin Med 1958, 46, 860-865), thin layerchromatography (Fausa O, and Shalhegg B A. 1976 Quantitativedetermination of bile acids and their conjugates using thin-layerchromatography and a purified 3-α hydroxysteroid dehydrogenase. Scand JGastroenterol 9, 249-254.), high performance liquid chromatography(Islam S, et al Fasting serum bile acid level in cirrhosis “A semiquantitative index of hepatic function”. J Hepatol 1985, 1, 609-617;Paauw J D, at al. Assay for taurine conjugates of bile acids in serum byreversed phase high performance liquid chromatography. J Chromatograph BBiomed Appl 1996, 685, 171-175), radioimmunoassay (Wildgrube J,Stockhausan H, Peter M, Mauritz G, Mandawi R. Radioimmunoassay of bileacids in tissues, bile and urine. Clin Chem 1983, 29, 494-498), enzymelinked colorimetric and radioimmunoassay (Guo W, et al. A study ondetection of serum fasting total bile acid and chologlycin in neonatesfor cholestasis. Chin Med Sci J 1996, 11, 244-247.), mass spectrometry(Sjovell J, et al. Mass spectrometry of bile acids. Method inenzymology. Vol. III, Academic Press, New York 1985.), tandem massspectrometry (Griffiths W J. Tandem mass spectrometry in study of fattyacids, bile acids and steroids. Mass Spectrum Rev 2003, 22, 81-152.),gas chromatography using high resolution glass capillary columns andmass spectrometry (Setchell K D R, Matsui A. Serum bile acid analysis.Clin Chim Acta 1983, 127, 1-17.), gas chromatography (Fischer S, et al.Hepatic levels of bile acids in end stage chronic cholestatic liverdisease. Clin Chim Acta 1996, 251, 173-186.), gas liquid chromatography(Van Berge Hengouwen G P et al., Quantitative anaylsis of bile acids inserum and bile, using gas liquid chromatography. Clin Chim Acta 1974,54, 249-261; Batta A K, et al. Characterization of serum and urinarybile acids in patients with primary biliary cirrhosis by gas-liquidchromatography-mass spectrometry: effect of ursodeoxycholic acidtreatment. J Lipid Res 1989, 30, 1953-1962), luminometric method(Styrellius I, Thore A, Bjorkhem I. Luminometric method. In: Methods ofenzymatic analysis. (Ed. III). Bergmeyer, Hans Ulirch [Hrsg]. 8:274-281, 1985.), UV method for bile assay (Stayer E, et al. Fluorimetricmethod for serum. In: Methods of enzymatic analysis. (Ed.III).Bergmeyer, Hans Ulrich; [Hrsg]. 8, 288-290, 1985; Stayer E, et al. UVmethod for bile, gastric juice and duodenai aspirates. In: Methods ofenzymatic analysis, (e.d.III). Bergmeyer, Hans Ulrich [Hrsg]. 8:285-287, 1985), enzymatic colorimetric method (Collins B J, et al.Measurement of total bile acids in gastric juice. J Clin Pathol 1984,37, 313-316) and enzymatic fluorimetric method can be used (Murphy G M,et al. A fluorometric and enzymatic method for the estimation of serumtotal bile acids. J Clin Path 1970, 23, 594-598; Hanson N Q, Freier E F.Enzymic measurement of total bile acid adapted to the Cobas FaraCentrifugal analyzer. Clin Chem. 1985, 35, 1538-1539).

Importance of Short Chain Fatty Acids (SCFA) to Human Health and Role ofMicrobiota

Short chain fatty acids (SCFAs) are a principal product of bacterialfermentation in the colon. SCFAs, particularly acetate, propionate andbutyrate, are thought to have many potential benefits to the mammalianhost. SCFAs are organic acids with fewer than 6 carbons and includeacetate, propionate, butyrate, valerate, isovalerate, and 2-methylbutyrate. While longer chain fatty acids are derived primarily fromdietary sources, SCFAs are derived from the breakdown of non-digestibleplant fiber. Butyrate is a primary energy source for colonocytes,whereas propionate is thought to be metabolized mostly by the liver viaportal vein circulation from the colon. Acetate is derived from themicrobiota is thought to be more generally available to tissues.

In addition to acting as metabolic substrates, SCFAs have multiplebenefits, including that SCFAs produced by the microbiota are essentialfor immune homeostasis and particularly for immune modulation byregulatory T cells. Direct ingestion of acetate, propionate or butyrate,or a mixture of all three by mice, stimulates the proliferation andmaturation of regulatory T cells (Tregs) that reside in the colon. Micegiven SCFAs in drinking water have significantly higher levels ofcolonic CD4+ FoxP3+ T cells (Tregs) than germ free and SPFA controls,and these Treg cells are functionally more potent as measured by theexpression of IL-10 mRNA and protein, and by their ability to inhibitCD8+ effector T cells in vitro (Smith P M et al. 2013 Science Themicrobial metabolites, short-chain fatty acid regulate colonic Treg cellhomeostasis 341: 569-73). This effect of SCFAs is mediated via signalingthrough GPR43 (FFAR2), a G protein coupled receptor expressed on avariety of cells but with high frequency on colonic Treg cells. GPR43signaling is upstream of modification of histone deacetylase activity(particularly HDAC9 and HDCA3), which is known to alter gene expressionvia reconfiguration of chromatin. Furthermore, the effects ofexperimental colitis induced by adoptive T cell transfer are reduced bySCFAs including propionate alone and a mixture of acetate, propionate orbutyrate in a GPR43 dependent fashion.

Beyond the direct effects of SCFA administered orally to animals,microbes can produce SCFA in situ in the colon and improve outcomes inseveral disease models. Daily administration of 10⁹ cfu ofButyricicoccus pullicaecorum, a butyrate forming organism first isolatedfrom chickens, for 1 week ameliorates TNBS-induced colitis in a ratmodel (Eeckhaut V et al., 2013 Gut Progress towards butyrate-producingpharmabiotics: Butyricicoccus pullicaecorum capsule and efficacy in TNBSmodels in comparison with therapeutics doi:10.1136/gutjnl-2013-305293).In humans, topical administration of butyrate or sodium butyrate via arectal enema may be beneficial to ulcerative colitis patients (ScheppachW et al. 1992 Gastroenterol Effect of butyrate enemas on the colonicmucosa in distal ulcerative colitis 103: 51-56; Vernia P et al. 2003Eur. J. Clin. Investig Topical butyrate improves efficacy of 5-ASA inrefractory distal ulcerative colitis: results of a multicentre trial.33: 244-48). Butyrate has effects at multiple levels including signalingvia GPR109A, which is expressed on the apical surface of intestinalepithelial cells (IECs). GPR109A lowers NFKB-mediated gene expression,including reduced expression of the inflammatory cytokines TNF-alpha,IL-6 and IL-1beta.

Oral administration of SCFAs in mice also has direct effects onmetabolism. SCFAs are a significant energy source and thus fermentationby the microbiota can contribute up 5-10% of the basal energyrequirements of a human. SCFAs upregulate production of glucagon-likepeptide 1 (GLP-1), peptide (P)YY and insulin. GLP-1 and PYY are noted toplay a role in enhancing satiety and reducing food intake. Furthermore,fecal transplantation from lean human donors to obese recipients withmetabolic syndrome results in a significant increase in insulinsensitivity after 6 weeks. This change is most correlated with thetransfer of Eubacterium hallii, a gram-positive, butyrate-fermentingmicrobe (Vrieze, A., et al., 2012 Gastroenterol Transfer of intestinalmicrobiota from lean donors increases insulin sensitivity 143: 913-6).

A common factor underlying both diabetes and obesity is the phenomenonof low-level inflammation termed metabolic endotoxemia (see below).Metabolic endotoxemia refers to increased permeability of the gut(“leaky gut syndrome”) coupled with increased translocation oflipopolysaccharide (LPS), mediating an inflammatory response thattriggers insulin resistance, changes in lipid metabolism, and liverinflammation responsible for non-alcoholic fatty liver disease (NAFLD).Low level bacteremia may also lead to the translocation of viablegram-negative organisms into distal tissues, such as adipocytes, andfurther drive inflammation. SCFAs provide a benefit here as well, bothby providing an energy source to enhance colonic epithelial cellintegrity and by stimulating the expression of tight junction proteinsto reduce translocation of gram-negative LPS, bacterial cells and theirfragments (Wang H B et al, 2012 Dig Dis Sci Butyrate enhances intestinalepithelial barrier function via up-regulation of tight junction proteinClaudin-1 transcription 57: 3126-35).

For all of these reasons, it would be useful to have microbialcommunities with an enhanced ability to produce SCFAs for the treatmentof diseases such as diabetes, obesity, inflammation, ulcerative colitisand NAFLD.

Acetate, propionate and butyrate are formed as end-products in anaerobicfermentation. SCFA producing bacteria in the gut gain energy bysubstrate-level phosphorylation during oxidative breakdown of carbonprecursors. However, the resulting reducing equivalents, captured in theform of NADH, must be removed to maintain redox balance, and hence theenergetic driving force to produce large amounts of reduced end-productssuch as butyrate and propionate, in order to regenerates NAD+. Acetate,propionate and butyrate are not the only end products of fermentation:microbes in the gut also produce lactate, formate, hydrogen and carbondioxide depending on the conditions. As discussed below, lactate andacetate can also drive the formation of butyrate and propionate throughcross-feeding by one microorganism to another.

The rate of SCFA production in the colon is highly dependent on manyfactors including the availability of polysaccharide carbon sources(such as, but not limited to, fructans, starches, cellulose,galactomannans, xylans, arabinoxylans, pectins, inulin,fructooligosaccharides, and the like), the presence of alternativeelectron sinks such as sulfate and nitrate, the redox potential,hydrogen (H₂) partial pressure and pH. As described above, cross-feedingamong organisms can also play a role, for instance when a lactateforming organism provides lactate as a substrate for a butyrate orpropionate producer, or when a saccharolytic organism breaks down acomplex carbohydrate to provide a mono- or disaccharide forfermentation. Acetate, which can be as high as 30 mM in the gut, is alsoa key building block of butyrate through the action of the enzymebutyryl-CoA:acetate CoA transferase, the final step in the production ofbutyrate. Importantly, this enzyme can also function as a propionyl-CoA:acetate CoA transferase, resulting in the production of propionate.

Since diet is a principal determinant of the variety of carbon sourcesand other nutrients available in the colon, it is clear that afunctional ecology for SCFA production will comprise multiple organismscapable of adapting to diet-driven changes in in the gut environment.Thus, there exists a need for a bacterial composition that can fermentsufficient quantities of SCFA products in spite of the varyingenvironmental conditions imposed by a changing diet. Such bacterialcompositions will comprise organisms capable of fermenting a variety ofcarbon sources into SCFA.

Role of Microbiota in Metabolic Endotoxemia/Bacteremia

Chronic, low-grade inflammation is characteristic of obesity and isrecognized to play an underlying pathogenic role in the metaboliccomplications and negative health outcomes of the disease. Notably,obesity is associated with elevated plasma levels of bacteriallipopolysaccharide (LPS). Energy intake, in particular a high fat diet(HFD), increases gut permeability and increases plasma LPS levels 2- to3-fold. LPS in the circulatory system reflects passage of bacterialfragments across the gut into systemic circulation (termed “metabolicendotoxemia”), either through increases in diffusion due to intestinalparacellular permeability or through absorption by enterocytes duringchylomicron secretion. Subcutaneous infusion of LPS into wild type micemaintained on a normal chow diet for 4 weeks leads to increased wholebody, liver and adipose tissue weights, adipose and liver inflammationas well as fasted hyperglycemia and insulinemia, effects that arecomparable to those induced by HFD (Cani et al., 2007 Diabetes.Metabolic endotoxemia initiates obesity and insulin resistancedoi:10.2337/db06-1491). In addition to bacterial fragments, thetranslocation of live bacteria to host tissues may also be a feature ofobesity (termed “metabolic bacteremia”) (Shen et al., 2013 Mol AspectsMed. The gut microbiota, obesity and insulin resistance doi:10.1016/j.mam.2012.11.001).

Host-microbiota interactions at the gut mucosal interface are involvedin intestinal barrier functionality and bacterialsurveillance/detection. Dysbiosis can promote bacterial translocationand obesity-associated inflammation. In one instance, metabolicendotoxemia of HFD-induced obesity in mice is associated with reductionsin Bifidobacterium, and both may be ameliorated through treatment withinulin (oligofructose) (Cani et al. Diabetologia Selective increases ofbifidobacteria in gut microflora improve high-fat-diet-induced diabetesin mice through a mechanism associated with endotoxaemia doi:10.1007/s00125-007-0791-0). The beneficial effects of inulin andBifidobacterium are associated with enhanced production ofintestinotrophic proglucagon-derived peptide 2 (GLP-2), a peptideproduced by L cells of the intestine that promotes intestinal growth(Cani et al. Gut. Changes in gut microbiota control inflammation inobese mice through a mechanism involving GLP-2-driven improvement of gutpermeability. doi: 10.1136/gut.2008.165886). Alternative pathwaysinvolving host-microbiota interactions and intestinal barrier integrityand metabolic endotoxemia/bacteremia include but are not limited tothose involving intestinotrophic proglucagon-derived peptide (GLP)-2,endocannabinoid (eCB) signaling, and pattern recognition receptorsincluding nucleotide-binding oligomerization domain (NOD)-like receptors(NLR) such as NOD1/NLRC1 and NOD2/NLRC2 as well as Toll like receptors(TLR) such as TLR-2, TLR-4, TLR-5 and TLR adapter protein myeloiddifferentiation primary-response protein 88 (MyD88) (see review Shen etal., 2013 Mol Aspects Med. The gut microbiota, obesity and insulinresistance doi: 10.1016/j.mam.2012.11.001).

Role of Microbiota in Energy Harvesting and Storage

The gut microbiota is involved in host energy harvesting. Germ free (GF)mice consume more energy but are significantly leaner than wild typecounterparts. Conventionalization of GF mice given a low-fat,polysaccharide-rich diet with the microbiota of conventionally-raisedmice leads to 60% more adiposity and insulin resistance despite reducedfood intake (Backhed et al., 2004 PNAS The gut microbiota as anenvironmental factor that regulates fat storage doi:10.1073/pnas.0407076101). GF mice conventionalized with microbiota fromobese mice show significantly greater increase in total body fat than GFmice conventionalized with microbiota from lean mice. Obese (ob/ob) micehave significantly less energy remaining in their feces relative totheir lean littermates as measured by bomb calorimetry (Turnbaugh et al.2006 Nature. An obesity-associated gut microbiome with increasedcapacity for energy harvest doi: 10.1038/nature05414). In humans,“overnutrition” (defined as energy consumption as a percentage ofweight-maintaining energy needs) is associated with proportionally moreFirmicutes and fewer Bacteroidetes and energy loss (stool calories as apercentage of ingested calories) in lean subjects is negativelyassociated with the proportional change in Firmicutes and positivelyassociated with the proportional change in Bacteroidetes, suggestingimpact of the gut microbiota on host energy harvest (Jumpertz et al.,2011 Am J Clin Nutr. Energy-balance studies reveal associations betweengut microbes, caloric load, and nutrient absorption in humans. doi:10.3945/ajcn.110.010132).

In addition to affecting host energy harvesting, gut microbiota is alsoimplicated in energy storage. The increase in body fat observed uponconventionalization of GF mice is associated with a decrease inFasting-induced adipose factor (Fiaf) expression in the ileum and a 122%increase in Lipoprotein Lipase (LPL) activity in epididymal adiposetissue (Backhed et al., 2004 PNAS The gut microbiota as an environmentalfactor that regulates fat storage doi/10.1073/pnas.0407076101). Fiaf(also known as angiopoietin-like 4) is a protein secreted by adiposetissues, liver and intestine that inhibits the activity of LPL, a keyenzyme in the hydrolysis of lipoprotein-associated triglycerides and therelease of fatty acids for transport into cells. In adipocytes, fattyacids released by LPL are re-esterified into triglyceride and stored asfat (Shen et al., 2013 Mol Aspects Med. The gut microbiota, obesity andinsulin resistance doi: 10.1016/j.mam.2012.11.001).

Other Functional Pathways

The pathways and mechanisms discussed above on the functional pathwaysand mechanisms by which the microbiota shape host health and disease isnot meant to be exhaustive. Alternative functional pathways andmechanisms exist, including but not limited to pathways involvingAMP-activated protein kinase (AMPK), TLR-5, and SREBP-1c and ChREBP.

Emergence of Antibiotic Resistance in Bacteria

Antibiotic resistance is an emerging public health issue (Carlet J,Collignon P, Goldmann D, Goossens H, Gyssens I C, Harbarth S, Jarlier V,Levy S B, N′Doye B, Pittet D, et al. 2011. Society's failure to protecta precious resource: antibiotics. Lancet 378: 369-371.). Numerous generaof bacteria harbor species that are developing resistance toantibiotics. These include but are not limited to Vancomycin ResistantEnterococcus (VRE) and Carbapenem resistant Klebsiella (CRKp).Klebsiella pneumoniae and Escherichia coli strains are becomingresistant to carbapenems and require the use of old antibioticscharacterized by high toxicity, such as colistin (Canton R, Akóva M,Carmeli Y, Giske C G, Glupczynski Y, Gniadkowski M, Livermore D M,Miriagou V, Naas T, Rossolini G M, et al. 2012. Rapid evolution andspread of carbapenemases among Enterobacteriaceae in Europe. ClinMicrobiol Infect 18: 413-431.). Further multiply drug resistant strainsof multiple species, including Pseudomonas aeruginosa, Enterobacter spp,and Acinetobacter spp are observed clinically including isolates thatare highly resistant to ceftazidime, carbapenems, and quinolones(European Centre for Disease Prevention and Control: EARSS net database.ecdc.europa.eu). The Centers for Disease Control and Prevention in 2013released a Threat Report (cdc.gov/drugresistance/threat_report 2013)citing numerous bacterial infection threats that included Clostridiumdifficile, Carbapenem-resistant Enterobacteriaceae (CRE),Multidrug-resistant Acinetobacter, Drug-resistant Campylobacter,Extended spectrum β-lactamase producing Enterobacteriaceae (ESBLs),Vancomycin-resistant Enterococcus (VRE), Multidrug-resistant Pseudomonasaeruginosa, Drug-resistant Non-typhoidal Salmonella, Drug-resistantSalmonella Typhi, Drug-resistant Shigella, Methicillin-resistantStaphylococcus aureus (MRSA), Drug-resistant Streptococcus pneumoniae,Vancomycin-resistant Staphylococcus aureus (VRSA),Erythromycin-resistant Group A Streptococcus, and Clindamycin-resistantGroup B Streptococcus. The increasing failure of antibiotics due therise of resistant microbes demands new therapeutics to treat bacterialinfections. Administration of a microbiome therapeutic bacterialcomposition offers potential for such therapies. Applicants havediscovered that patients suffering from recurrent C. difficileassociated diarrhea (CDAD) often harbor antibiotic resistantGram-negative bacteria, in particular Enterobacteriaceae and thattreatment with a bacterial composition effectively treats CDAD andreduces the antibiotic resistant Gram-negative bacteria. Thegastrointestinal tract is implicated as a reservoir for many of theseorganisms including VRE, MRSA, Pseudomonas aeruginosa, Acinetobacter andthe yeast Candida (Donskey, Clinical Infectious Diseases 2004 39:214,TheRole of the Intestinal Tract as a Reservoir and Source for Transmissionof Nosocomial Pathogens), and thus as a source of nosocomial infections.Antibiotic treatment and other decontamination procedures are among thetools in use to reduce colonization of these organisms in susceptiblepatients including those who are immunosuppressed. Bacterial-basedtherapeutics would provide a new tool for decolonization, with a keybenefit of not promoting antibiotic resistance as antibiotic therapiesdo.

Compositions of the Invention

Network Ecologies

As described above, the Network Ecology and Functional Network Ecologyrefer to a consortium of OTUs or Functional modalities respectively thatco-occur in a group of subjects. The network is defined mathematicallyby a graph delineating how specific nodes (i.e., OTUs or functionalmodalities) and edges (connections between specific OTUs or functionalmodalities) relate to one another to define the structural ecology of aconsortium of OTUs or functions. Any given Network Ecology or FunctionalNetwork Ecology will possess inherent phylogenetic diversity andfunctional properties.

A Network Class or Core Network refers to a group of Network Ecologiesor Functional Network ecologies that are computationally determined tocomprise ecologies with similar phylogenetic and/or functionalcharacteristics. A Network Class or Core Network therefore containsimportant biological features, defined either phylogenetically orfunctionally, of a group (i.e., a cluster) of related network ecologies.

Keystone OTUs or Functions are one or more OTUs or Functions that arecommon to many network Ecologies or Functional Netwok Ecologies and aremembers of Networks Ecologies or Functional Network Ecologies that occurin many subjects (i.e., are pervasive). Due to the ubiquitous nature ofKeystone OTUs and Functions, they are central to the function of networkecologies in healthy subjects and are often missing or at reduced levelsin subjects with disease. Keystone OTUs and Functions may exist in low,moderate, or high abundance in subjects.

Bacteria that are members of the keystone OTUs, core network or networkecology are provided herein.

Bacterial Compositions

Provided are bacteria and combinations of bacteria that comprise networkecologies and functional network ecologies of the human gut microbiota.The bacteria and combinations of bacteria that comprise networkecologies have a capacity to meaningfully provide functions of a healthymicrobiota when administered to mammalian hosts. Without being limitedto a specific mechanism, it is believed that the members of networkecologies can inhibit the growth, proliferation, germination and/orcolonization of one or a plurality of pathogenic bacteria in thedysbiotic microbiotal niche, and may also augment the growth,proliferation, germination and/or colonization of desired bacteria sothat a healthy, diverse and protective microbiota colonizes andpopulates the intestinal lumen to establish or reestablish ecologicalcontrol over pathogens or potential pathogens (e.g., some bacteria arepathogenic bacteria only when present in a dysbiotic environment). Theterm pathogens refers to a bacterium or a group of bacteria or any otherorganism or entity that is capable of causing or affecting a disease,disorder or condition of a host containing the bacterium, organism orentity, including but not limited to metabolic diseases such asprediabetes, type 1 diabetes, and type 2 diabetes.

As used herein, a “type” or more than one “types” of bacteria may bedifferentiated at the genus level, the species, level, the sub-specieslevel, the strain level or by any other taxonomic method, as describedherein and otherwise known in the art.

Bacterial compositions can comprise two types of bacteria (termed“binary combinations” or “binary pairs”), and typically a large numberof bacteria types. For instance, a bacterial composition can comprise atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, or at least 21, 22, 23, 24, 25, 26, 27, 28, 2930, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at least 40, at least 50 orgreater than 50 types of bacteria, as defined by species or operationaltaxonomic unit (OTU), or otherwise as provided herein. In someembodiments, the bacterial composition includes at least 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or greaternumbers of bacteria types.

In another embodiment, the number of types of bacteria present in abacterial composition is at or below a known value. For example, in suchembodiments the network ecology comprises 1000, 900, 800, 700, 600, 500,400, 300, 200, 100, 50 or fewer types of bacteria, such as 49, 48, 47,46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,or 10 or fewer, or 9 or fewer types of bacteria, 8 or fewer types ofbacteria, 7 or fewer types of bacteria, 6 or fewer types of bacteria, 5or fewer types of bacteria, 4 or fewer types of bacteria, or 3 or fewertypes of bacteria.

Bacterial Compositions Described by Species

Bacterial compositions that comprise network ecologies may be preparedcomprising at least two types of isolated bacteria, chosen from thespecies in Table 1.

In one embodiment, the bacterial composition that comprises at least oneand preferably more than one of the following: Enterococcus faecalis(previously known as Streptococcus faecalis), Clostridium innocuum,Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus,Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1),Clostridum bifermentans, and Blautia producta (previously known asPeptostreptococcus productus). In an alternative embodiment, at leastone of the preceding species is not substantially present in thebacterial composition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Enterococcus faecalis(previously known as Streptococcus faecalis), Clostridium innocuum,Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus,Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1),Clostridum bifermentans, and Blautia producta (previously known asPeptostreptococcus productus). In an alternative embodiment, at leastone of the preceding species is not substantially present in thebacterial composition.

In another embodiment, the bacterial composition comprises at least oneand preferably more than one of the following: Acidaminococcusintestinalis, Bacteroides ovatus, two strains of Bifidobacteriumadolescentis, two strains of Bifidobacterium longum, Blautia producta,Clostridium cocleatum, Collinsella aerofaciens, two strains of Dorealongicatena, Escherichia coli, Eubacterium desmolans, Eubacteriumeligens, Eubacterium limosum, four strains of Eubacterium rectale,Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lachnospirapectinoshiza, Lactobacillus casei, Lactobacillus casei/paracasei,Paracateroides distasonis, Raoultella sp., one strain of Roseburia(chosen from Roseburia faecalis or Roseburia faecis), Roseburiaintestinalis, two strains of Ruminococcus torques, two strains ofRuminococcus obeum, and Streptococcus mitis. In an alternativeembodiment, at least one of the preceding species is not substantiallypresent in the bacterial composition.

In yet another embodiment, the bacterial composition comprises at leastone and preferably more than one of the following: Barnesiellaintestinihominis; Lactobacillus reuteri; a species characterized as oneof Enterococcus hirae, Enterococus faecium, or Enterococcus durans; aspecies characterized as one of Anaerostipes caccae or Clostridiumindolis; a species characterized as one of Staphylococcus warneri orStaphylococcus pasteuri; and Adlercreutzia equolifaciens. In analternative embodiment, at least one of the preceding species is notsubstantially present in the bacterial composition.

In other embodiments, the bacterial composition comprises at least oneand preferably more than one of the following: Clostridium absonum,Clostridium argentinense, Clostridium baratii, Clostridium bartlettii,Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum,Clostridium cadaveris, Clostridium camis, Clostridium celatum,Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium difficile, Clostridium fallax, Clostridiumfelsineum, Clostridium ghonii, Clostridium glycolicum, Clostridiumhaemolyticum, Clostridium hastiforme, Clostridium histolyticum,Clostridium indolis, Clostridium innocuum, Clostridium irregulare,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense,Clostridium sartagoforme, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum,Clostridium tertium, Clostridium tetani, Clostridium welchii, andClostridium villosum. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Clostridium innocuum, Clostridum bifermentans, Clostridiumbutyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides uniformis, three strains of Escherichia coli, andLactobacillus sp. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Clostridium bifermentans, Clostridium innocuum, Clostridiumbutyricum, three strains of Escherichia coli, three strains ofBacteroides, and Blautia producta. In an alternative embodiment, atleast one of the preceding species is not substantially present in thebacterial composition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Bacteroides sp., Escherichia coli, and non pathogenicClostridia, including Clostridium innocuum, Clostridium bifermentans andClostridium ramosum. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Bacteroides species, Escherichia coli and non-pathogenicClostridia, such as Clostridium butyricum, Clostridium bifermentans andClostridium innocuum. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Bacteroides caccae, Bacteroides capillosus, Bacteroidescoagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroidesforsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroidesgracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae,Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis,Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris,Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides ureolyticus, and Bacteroides vulgatus. In an alternativeembodiment, at least one of the preceding species is not substantiallypresent in the bacterial composition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Bacteroides, Eubacteria, Fusobacteria, Propionibacteria,Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger,Desulfomonas, and Peptostreptococcus. In an alternative embodiment, atleast one of the preceding species is not substantially present in thebacterial composition.

In one embodiment, the bacterial composition that comprises a networkecology comprises at least one and preferably more than one of thefollowing: Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens,Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previouslyknown as Peptostreptococcus productus II), Bacteroides fragilis ss.Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacteriumaerofaciens III, Blautia producta (previously known asPeptostreptococcus productus I), Ruminococcus bronii, Bifidobacteriumadolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacteriumsiraeum, Ruminococcus torques, Eubacterium rectale Eubacterium rectaleIV, Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum,Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacteriuminfantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroidescapillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacteriumhallii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcusobeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillusleichmanii, Ruminococcus cailidus, Butyrivibrio crossotus,Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilisss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum,Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1,Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum,Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferumFusobacterium naviforme, Clostridium innocuum, Clostridium ramosum,Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT,Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroidesfragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacteriumnucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcusmorbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcusintermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X,Coprococcus BH, —CC; Eubacterium tenue, Eubacterium ramulus, EubacteriumAE, -AG-H, -AG-M, AJ, -BN-1; Bacteroides clostridiiformis ss.clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroidesruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus,Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H,Lactobacillus G, and Succinivibrio A. In an alternative embodiment, atleast one of the preceding species is not substantially present in thebacterial composition.

Bacterial Compositions Described by Operational Taxonomic Unit (OTUs)

Bacterial compositions may be prepared comprising at least two types ofisolated bacteria, chosen from the SEQ ID Numbers (OTUs) in Table 1.

OTUs can be defined either by full 16S sequencing of the rRNA gene(Table 1), by sequencing of a specific hypervariable region of this gene(i.e. V1, V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing of anycombination of hypervariable regions from this gene (e.g. V1-3 or V3-5).The bacterial 16S rDNA is approximately 1500 nucleotides in length andis used in reconstructing the evolutionary relationships and sequencesimilarity of one bacterial isolate to another using phylogeneticapproaches. 16S sequences are used for phylogenetic reconstruction asthey are in general highly conserved, but contain specific hypervariableregions that harbor sufficient nucleotide diversity to differentiategenera and species of most microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S sequence, genomicDNA is extracted from a bacterial sample, the 16S rDNA (full region orspecific hypervariable regions) amplified using polymerase chainreaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingcan be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

OTUs can be defined by a combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof, full-genome sequence, or partial genome sequencegenerated using amplified genetic products, or whole genome sequence(WGS). Using well defined methods familiar to one with ordinary skill inthe art, DNA extracted from a bacterial sample will have specificgenomic regions amplified using PCR and sequenced to determine thenucleotide sequence of the amplified products. In the whole genomeshotgun (WGS) method, extracted DNA will be directly sequenced withoutamplification. Sequence data can be generated using any sequencingtechnology including, but not limited to Sanger, Illumina, 454 LifeSciences, Ion Torrent, ABI, Pacific Biosciences, and/or Oxford Nanopore.

In one embodiment, the OTUs can be characterized by one or more of thevariable regions of the 16S sequence (V1-V9). These regions in bacteriaare defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879,986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. (See, e.g.,Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA genefrom Escherichia coli, PNAS 75(10):4801-4805 (1978)). In someembodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9regions are used to characterize an OTU. In one embodiment, the V1, V2,and V3 regions are used to characterize an OTU. In another embodiment,the V3, V4, and V5 regions are used to characterize an OTU. In anotherembodiment, the V4 region is used to characterize an OTU.

Bacterial Compositions Exclusive of Certain Bacterial Species Or Strains

In one embodiment, the bacterial composition does not comprise at leastone of Enterococcus faecalis (previously known as Streptococcusfaecalis), Clostridium innocuum, Clostridium ramosum, Bacteroidesovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichiacoli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta(previously known as Peptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise atleast one of Acidaminococcus intestinalis, Bacteroides ovatus, twospecies of Bifidobacterium adolescentis, two species of Bifidobacteriumlongum, Collinsella aerofaciens, two species of Dorea longicatena,Escherichia coli, Eubacterium eligens, Eubacterium limosum, four speciesof Eubacterium rectale, Eubacterium ventriosumi, Faecalibacteriumprausnitzii, Lactobacillus casei, Lactobacillus paracasei,Paracateroides distasonis, Raoultella sp., one species of Roseburia(chosen from Roseburia faecalis or Roseburia faecis), Roseburiaintestinalis, two species of Ruminococcus torques, and Streptococcusmitis.

In yet another embodiment, the bacterial composition does not compriseat least one of Barnesiella intestinihominis; Lactobacillus reuteri; aspecies characterized as one of Enterococcus hirae, Enterococus faecium,or Enterococcus durans; a species characterized as one of Anaerostipescaccae or Clostridium indolis; a species characterized as one ofStaphylococcus warneri or Staphylococcus pasteuri; and Adlercreutziaequolifaciens.

In other embodiments, the bacterial composition does not comprise atleast one of Clostridium absonum, Clostridium argentinense, Clostridiumbaratii, Clostridium bifermentans, Clostridium botulinum, Clostridiumbutyricum, Clostridium cadaveris, Clostridium camis, Clostridiumcelatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium difficile, Clostridium fallax, Clostridiumfelsineum, Clostridium ghonii, Clostridium glycolicum, Clostridiumhaemolyticum, Clostridium hastiforme, Clostridium histolyticum,Clostridium indolis, Clostridium innocuum, Clostridium irregulare,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense,Clostridium sartagoforme, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum,Clostridium tertium, Clostridium tetani, Clostridium welchii, andClostridium villosum.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium innocuum, Clostridum bifermentans, Clostridiumbutyricum, Bacteroides Bacteroides thetaiotaomicron, Bacteroidesuniformis, three strains of Escherichia coli, and Lactobacillus sp.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium bifermentans, Clostridium innocuum, Clostridiumbutyricum, three strains of Escherichia coli, three strains ofBacteroides, and Blautia producta (previously known asPeptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides sp., Escherichia coli, and non pathogenicClostridia, including Clostridium innocuum, Clostridium bifermentans andClostridium ramosum.

In another embodiment, the bacterial composition does not comprise atleast one of more than one Bacteroides species, Escherichia coli andnon-pathogenic Clostridia, such as Clostridium butyricum, Clostridiumbifermentans and Clostridium innocuum.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides caccae, Bacteroides capillosus, Bacteroidescoagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroidesforsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroidesgracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae,Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis,Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris,Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides ureolyticus, and Bacteroides vulgatus.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides, Eubacteria, Fusobacteria, Propionibacteria,Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger,Desulfomonas, and Peptostreptococcus.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens,Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previouslyknown as Peptostreptococcus productus II), Bacteroides fragilis ss.Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacteriumaerofaciens III, Blautia producta (previously known asPeptostreptococcus productus I), Ruminococcus bromii, Bifidobacteriumadolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacteriumsiraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacteriumrectale IV Eubacterium eligens, Bacteroides eggerthii, Clostridiumleptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacteriuminfantis, Eubacterium rectale III-F; Coprococcus comes, Bacteroidescapillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacteriumhallii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcusobeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillusleichmanii, Ruminococcus cailidus, Butyrivibrio crossotus,Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilisss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum,Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1,Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum,Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I,Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum,Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT,Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, AN; Bacteroidesfragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacteriumnucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcusmorbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcusintermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X,Coprococcus BH, —CC; Eubacterium tenue, Eubacterium ramulus, EubacteriumAE, -AG-H, -AG-M, AJ, -BN-1; Bacteroides clostridiiformis ss.clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroidesruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus,Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H,Lactobacillus G, and Succinivibrio A.

Inhibition of Bacterial Pathogens

In some embodiments, the bacterial composition provides a protective ortherapeutic effect against infection by one or more GI pathogens ofinterest. Table 1 provides a list of OTUs that are either pathogens,pathobionts, or opportunistic pathogens.

In some embodiments, the pathogenic bacterium is selected from the groupconsisting of Yersinia, Vibrio, Treponema, Streptococcus,Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas,Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella,Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia,Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium,Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella,Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug resistantbacteria, extended spectrum beta-lactam resistant Enterococci (ESBL),Carbapenem-resistent Enterobacteriaceae (CRE), and vancomycin-resistantEnterococci (VRE).

In some embodiments, these pathogens include, but are not limited to,Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides,Bacillus cereus, Campylobacter jejuni, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, enteroaggregativeEscherichia coli, enterohemorrhagic Escherichia coli, enteroinvasiveEscherichia coli, enterotoxigenic Escherichia coli (such as, but notlimited to, LT and/or ST), Escherichia coli 0157:H7, Helicobacterpylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonasshigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi,Shigella spp., Staphylococcus spp., Staphylococcus aureus,vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae,Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica.

In one embodiment, the pathogen of interest is at least one pathogenchosen from Clostridium difficile, Salmonella spp., pathogenicEscherichia coli, vancomycin-resistant Enterococcus spp., and extendedspectrum beta-lactam resistant Enterococci (ESBL).

Purified Spore Populations

In some embodiments, the bacterial compositions comprise purified sporepopulations or a combination of a purified spore population with anon-spore population. Purified spore populations contain combinations ofcommensal bacteria of the human gut microbiota with the capacity tomeaningfully provide functions of a healthy microbiota when administeredto a mammalian subject. Without being limited to a specific mechanism,it is thought that such compositions inhibit the growth of a pathogensuch as C. difficile, Salmonella spp., enteropathogenic E. coli, andvancomycin-resistant Enterococcus spp., so that a healthy, diverse andprotective microbiota can be maintained or, in the case of pathogenicbacterial infections such as C. difficile infection, repopulate theintestinal lumen to reestablish ecological control over potentialpathogens. In some embodiments, yeast spores and other fungal spores arealso purified and selected for therapeutic use.

Disclosed herein are therapeutic and prophylactic compositionscontaining non-pathogenic, germination-competent bacterial spores, sporeforming organisms and non-spore forming organisms, for the prevention,control, and treatment of gastrointestinal diseases, disorders andconditions and for general nutritional health. These compositions areadvantageous in being suitable for safe administration to humans andother mammalian subjects and are efficacious in numerousgastrointestinal diseases, disorders and conditions and in generalnutritional health. While spore-based compositions are known, these aregenerally prepared according to various techniques such aslyophilization or spray-drying of liquid bacterial cultures, resultingin poor efficacy, instability, substantial variability and lack ofadequate safety.

It has now been found that populations of bacterial spores can beobtained from biological materials obtained from mammalian subjects,including humans. These populations are formulated into compositions asprovided herein, and administered to mammalian subjects using themethods as provided herein.

Provided herein are therapeutic bacterial compositions containing apurified population of bacterial spores, spore forming organisms andnon-spore forming organisms.

As used herein, the terms “purify”, “purified” and “purifying” refer tothe state of a population (e.g., a plurality of known or unknown amountand/or concentration) of desired bacterial spores or bacteria, that haveundergone one or more processes of purification, e.g., a selection or anenrichment of the desired bacterial spore, or alternatively a removal orreduction of residual habitat products as described herein. In someembodiments, a purified population has no detectable undesired activityor, alternatively, the level or amount of the undesired activity is ator below an acceptable level or amount. In other embodiments, a purifiedpopulation has an amount and/or concentration of desired bacterialspores or bacteria at or above an acceptable amount and/orconcentration. In other embodiments, the purified population ofbacterial spores or bacteria is enriched as compared to the startingmaterial (e.g., a fecal material liquid culture) from which thepopulation is obtained. This enrichment may be by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%,99.999%, 99.9999%, or greater than 99.9999% as compared to the startingmaterial.

In certain embodiments, the purified populations of bacterial sporeshave reduced or undetectable levels of one or more pathogenicactivities, such as toxicity, an infection of the mammalian recipientsubject, an immunomodulatory activity, an autoimmune response, ametabolic response, or an inflammatory response or a neurologicalresponse. Such a reduction in a pathogenic activity may be by 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to thestarting material. In other embodiments, the purified populations ofbacterial spores have reduced sensory components as compared to fecalmaterial, such as reduced odor, taste, appearance, and umami.

Provided are purified populations of bacterial spores or bacteria thatare substantially free of residual habitat products. In certainembodiments, this means that the bacterial spore or bacterialcomposition no longer contains a substantial amount of the biologicalmatter associated with the microbial community while living on or in thehuman or animal subject, and the purified population of spores may be100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of anycontamination of the biological matter associated with the microbialcommunity. Substantially free of residual habitat products may also meanthat the bacterial spore composition contains no detectable cells from ahuman or animal, and that only microbial cells are detectable, inparticular, only desired microbial cells are detectable. In anotherembodiment, it means that fewer than 1×10′%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%,1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸% of the cells in the bacterial composition arehuman or animal, as compared to microbial cells. In another embodiment,the residual habitat product present in the purified population isreduced at least a certain level from the fecal material obtained fromthe mammalian donor subject, e.g., reduced by at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, 99.9999%, or greater than 99.9999%.

In one embodiment, substantially free of residual habitat products orsubstantially free of a detectable level of a pathogenic material meansthat the bacterial composition contains no detectable viral (includingbacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmalcontaminants, or a eukaryotic parasite such as a helminth.Alternatively, the purified spore populations are substantially free ofan acellular material, e.g., DNA, viral coat material, or non-viablebacterial material.

As described herein, purified spore populations can be demonstrated bygenetic analysis (e.g., PCR, DNA sequencing), serology and antigenanalysis, and methods using instrumentation such as flow cytometry withreagents that distinguish desired bacterial spores from non-desired,contaminating materials.

Exemplary biological materials include fecal materials such as feces ormaterials isolated from the various segments of the small and largeintestines. Fecal materials are obtained from a mammalian donor subject,or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400,500, 750, 1000 or from greater than 1000 donors, where such materialsare then pooled prior to purification of the desired bacterial spores.

In alternative embodiments, the desired bacterial spores or bacteria arepurified from a single fecal material sample obtained from a singledonor, and after such purification are combined with purified sporepopulations or bacteria from other purifications, either from the samedonor at a different time, or from one or more different donors, orboth.

Preferred bacterial genera include Acetonema, Alkaliphilus,Alicyclobacillus, Amphibacillus, Ammonifex, Anaerobacter, Anaerofustis,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Blautia,Brevibacillus, Bryantella, Caldicellulosiruptor, Caloramator,Candidatus, Carboxydibrachium, Carboxydothermus, Clostridium, Cohnella,Coprococcus, Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Desulfotomaculum, Dorea, Eubacterium, Faecalibacterium, Filifactor,Geobacillus, Halobacteroides, Heliobacillus, Heliobacterium,Heliophilum, Heliorestis, Lachnoanaerobaculum, Lysinibacillus, Moorella,Oceanobacillus, Orenia (S.), Oxalophagus, Oxobacter, Paenibacillus,Pelospora, Pelotomaculum, Propionispora, Roseburia, Ruminococcus,Sarcina, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa,Sporosarcina, Sporotomaculum, Subdoligranulum, Symbiobacterium,Syntrophobotulus, Syntrophospora, Terribacillus, Thermoanaerobacter, andThermosinus.

In some embodiments, spore-forming bacteria are identified by thepresence of nucleic acid sequences that modulate sporulation. Inparticular, signature sporulation genes are highly conserved acrossmembers of distantly related genera including Clostridium and Bacillus.Traditional approaches of forward genetics have identified many, if notall, genes that are essential for sporulation (spo). The developmentalprogram of sporulation is governed in part by the successive action offour compartment-specific sigma factors (appearing in the order σF, σE,σG and σK), whose activities are confined to the forespore (σF and σG)or the mother cell (σE and σK).

Provided are spore populations containing more than one type ofbacterium. As used herein, a “type” or more than one “types” of bacteriamay be differentiated at the genus level, the species, level, thesub-species level, the strain level or by any other taxonomic method, asdescribed herein and otherwise known in the art.

In some embodiments, all or essentially all of the bacterial spores orbacterial species present in a purified population are originallyisolated obtained from a fecal material treated as described herein orotherwise known in the art. In alternative embodiments, one or more thanone bacterial spores, bacteria, or types of bacterial spores aregenerated in culture and combined to form a purified bacterialcomposition, including a purified spore population. In other alternativeembodiments, one or more of these culture-generated populations arecombined with a fecal material-derived populations to generate a hybridpopulation. Bacterial compositions may contain at least two types ofthese preferred bacteria, including strains of the same species. Forinstance, a bacterial composition may comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, or atleast 20 or more than 20 types of bacteria, as defined by species oroperational taxonomic unit (OTU) encompassing such species.

Thus, provided herein are methods for production of a bacterialcomposition containing a population of bacterial spores suitable and/ornon-sporulating bacteria for therapeutic administration to a mammaliansubject in need thereof. And the composition is produced by generallyfollowing the steps of: (a) providing a fecal material obtained from amammalian donor subject; and (b) subjecting the fecal material to atleast one purification treatment or step under conditions such that apopulation of bacterial spores is produced from the fecal material. Thecomposition is formulated such that a single oral dose contains at leastabout 1×10⁴ colony forming units of the bacterial spores, and a singleoral dose will typically contain about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷,1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greaterthan 1×10¹⁵ CFUs of the bacterial spores. The presence and/orconcentration of a given type of bacteria or bacterial spore may beknown or unknown in a given purified spore population. If known, forexample the concentration of bacteria or spores of a given strain, orthe aggregate of all strains, is e.g., 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷,1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greaterthan 1×10¹⁵ viable bacteria or bacterial spores per gram of compositionor per administered dose.

In some formulations, the composition contains at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% spores on a massbasis. In some formulations, the administered dose does not exceed 200,300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.

The bacterial compositions are generally formulated for oral or gastricadministration, typically to a mammalian subject. In particularembodiments, the composition is formulated for oral administration as asolid, semi-solid, gel, or liquid form, such as in the form of a pill,tablet, capsule, or lozenge. In some embodiments, such formulationscontain or are coated by an enteric coating to protect the bacteriathrough the stomach and small intestine, although spores are generallyresistant to the stomach and small intestines.

The bacterial compositions may be formulated to be effective in a givenmammalian subject in a single administration or over multipleadministrations. For example, a single administration is substantiallyeffective to reduce Cl. difficile and/or Cl. difficile toxin content ina mammalian subject to whom the composition is administered.Substantially effective means that Cl. difficile and/or Cl. difficiletoxin content in the subject is reduced by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% followingadministration of the composition.

Kits For Diagnosis of a State of Dysbiosis in a Subject

In some embodiments, the invention includes kits for carrying outmethods of the invention described herein and in the claims. In someembodiments, the invention includes a kit for diagnosis of a state ofdysbiosis in a mammalian subject in need thereof. In one embodiment, thekit includes a plurality of detection means suitable for use indetecting (1) a first bacterial entity comprising a keystone bacterialentity and (2) a second bacterial entity, wherein the first and secondbacterial entities comprise a Network Ecology, as described herein. Thekit can include instructions for use of the kit.

In other embodiments, the kit provides detection means, reagents, andinstructions for detecting a first bacterial entity and a secondbacterial entity that comprise a Network Ecology by: obtaining a fecalsample from a mammalian subject comprising a plurality of bacterialentities, contacting the fecal sample with a first detection moiety (andin some cases, a second detection moiety) capable of detecting the firstbacterial entity and the second bacterial entity present in the network,detecting the absence of the first and/or second bacterial entities inthe fecal sample, and thereby detecting a dysbiosis in the mammailiansubject. In some embodiments, the kit provides reagents and steps foradministering to the mammailian subject a composition comprising aneffect amount of the first and/or second bacterial species.

In some embodiments, the kit includes detection means and instructionsfor obtaining a fecal sample from the mammalian subject comprising aplurality of bacterial entities; contacting the fecal sample with afirst detection moiety capable of detecting a first bacterial entitypresent in an network; detecting the absence of the first bacterialentity in the fecal sample, thereby detecting a dysbiosis in themammalian subject; and administering to the mammalian subject acomposition comprising an effective amount of the first bacterialentity.

In other embodiments, the kit includes reagents and instructions for amethod for treating, preventing, or reducing the severity of a disorderselected from the group consisting of Clostridium difficile AssociatedDiarrhea (CDAD), Type 2 Diabetes, Obesity, Irritable Bowel Disease(IBD), colonization with a pathogen or pathobiont, and infection with adrug-resistant pathogen or pathobiont, comprising: administering to amammalian subject in need thereof an effective amount of a therapeuticbacterial composition, said therapeutic bacterial composition comprisinga plurality of isolated bacteria or a purified bacterial preparation,the plurality of isolated bacteria or the purified bacterial preparationcapable of forming a network ecology selected from the group consistingof those described throughout the specification.

In another embodiment, the kit includes reagents and instructions for amethod for producing short chain fatty acids (SCFA) within a mammaliansubject, comprising: administering to said mammalian subject in needthereof an effective amount of a therapeutic bacterial composition, saidtherapeutic bacterial composition comprising a plurality of isolatedbacteria or a purified bacterial preparation, the plurality of isolatedbacteria of the purified bacterial preparation capable of forming one ora plurality of bacterial functional pathways, the one or plurality ofbacterial functional pathways capable of forming a functional networkecology selected from the group consisting of those described throughoutthe specification.

In another embodiment, the kit includes reagents and instructions for amethod for catalyzing secondary metabolism of bile acids within amammalian subject, comprising: administering to said mammalian subjectin need thereof an effective amount of a therapeutic bacterialcomposition, said therapeutic bacterial composition comprising aplurality of isolated bacteria or a purified bacterial preparation, theplurality of isolated bacteria of the purified bacterial preparationcapable of forming one or a plurality of bacterial functional pathways,the one or plurality of bacterial functional pathways capable of forminga functional network ecology selected from the group consisting of thosedescribed throughout the specification.

Systems for Predicting a Dysbiosis in a Subject

The invention provides systems for predicting a dysbiosis in a subject,the system comprising: a storage memory for storing a dataset associatedwith a sample obtained from the subject, wherein the dataset comprisescontent data for at least one network of bacterial entities describedherein, and a processor communicatively coupled to the storage memoryfor determining a score with an interpretation function wherein thescore is predictive of dysbiosis in the subject.

In some embodiments, the invention provides systems for detecting adysbiosis in a subject comprising: a storage memory for storing adataset associated with a sample obtained from the subject, wherein thedataset comprises content data for at least one network of bacterialentities described herein, and a processor communicatively coupled tothe storage memory for determining a score with an interpretationfunction, wherein the score is predictive of dysbiosis in the subject.

An example of a computer system and its components that can be used toperform methods of the invention are described below in FIG. 21.

Computer Overview

FIG. 21 is a high-level block diagram illustrating an example of acomputer 2100 for use as a server or a user device, in accordance withone embodiment. Illustrated are at least one processor 2102 coupled to achipset 2104. The chipset 2104 includes a memory controller hub 2120 andan input/output (I/O) controller hub 2122. A memory 2106 and a graphicsadapter 2112 are coupled to the memory controller hub 2120, and adisplay device 2118 is coupled to the graphics adapter 2112. A storagedevice 2108, keyboard 2110, pointing device 2114, and network adapter2116 are coupled to the I/O controller hub 2122. Other embodiments ofthe computer 2100 have different architectures. For example, the memory2106 is directly coupled to the processor 2102 in some embodiments.

The storage device 2108 is a non-transitory computer-readable storagemedium such as a hard drive, compact disk read-only memory (CD-ROM),DVD, or a solid-state memory device. The memory 2106 holds instructionsand data used by the processor 2102. The pointing device 2114 is used incombination with the keyboard 2110 to input data into the computersystem 200. The graphics adapter 2112 displays images and otherinformation on the display device 2118. In some embodiments, the displaydevice 2118 includes a touch screen capability for receiving user inputand selections. The network adapter 2116 couples the computer system2100 to the network. Some embodiments of the computer 2100 havedifferent and/or other components than those shown in FIG. 21. Forexample, the server can be formed of multiple blade servers and lack adisplay device, keyboard, and other components.

The computer 2100 is adapted to execute computer program modules forproviding functionality described herein. As used herein, the term“module” refers to computer program instructions and other logic used toprovide the specified functionality. Thus, a module can be implementedin hardware, firmware, and/or software. In one embodiment, programmodules formed of executable computer program instructions are stored onthe storage device 2108, loaded into the memory 2106, and executed bythe processor 2102.

Methods of the Invention

Method of Determining Network Ecologies

Methods are provided for a computational approach based in part onnetwork theory to construct the ecology of a group of microorganismsbased on the presence or absence of specific OTUs (i.e., microbialgenera, species or strains) in a given set of sampled subjects. See FIG.16. See e.g., Cormen T H, Leiserson C E, Rivest R L, and Stein C. 2009.Introduction to Algorithms. Third edition. The MIT Press. Garey M R, andJohnson D S. 1979. Computers and Intractability: A Guide to the Theoryof NP-Completeness. First Edition. W. H. Freeman. The approach includesthe following: (i) identifying the microbial network ecologies that arepresent in both healthy and diseased subjects, (ii) identifying thekeystone OTUs and/or functions (FIG. 17), and phylogenetic clades thatcharacterize a given ecology, and (iii) providing specific metrics withwhich to prioritize the various network ecologies with respect to theircapacity to be useful in restoring the microbiome from a state ofdysbiosis to a state of health. In general the method first defines alllow and high order networks within given sets of subjects, and thenutilizes a comparative approach to define biologically significantnetworks.

This method comprises computing a co-occurrence matrix of OTUs (i.e.,presence or absence) for each subject across a defined subjectpopulation (populations are defined by a specific phenotype such as butnot limited to “subjects who are healthy”, or “subjects with disease”).The method comprises computing all nodes (OTUs, or species, or strains)and edges (co-occurrence between OTUs, or species, or strains) thatdefine the Network Ecology in a given subject's sample. Eachco-occurrence is scored using a discrete binary variable denotingpresence or absence. While the algorithm allows co-occurrences to beweighted based on the relative abundance of OTUs in the samples, ingeneral, this is undesirable since low abundance OTUs may be importantecologically. Furthermore, a discretized measure of presence or absenceof nodes eliminates bias and errors in the computed network ecologiesthat will arise from bias in methods used to generate relative abundancemeasures. A discreet method measuring presence or absence enables thedetection of low frequency OTUs and the elucidation of networks that areoften missed by methods based on relative abundance measures. Followingderivation of all low and high order networks in a given subject, onecan define all the network ecologies in a given phenotype (i.e.,collections of data sets from subjects with a unifying characteristic,for example, all data sets from healthy subjects) by defining the nodeand edge combinations that are maximally observed across all subjects.Without being bound by theory, it is understood that such networkecologies are present in a mammalian subject. The algorithm iterates theconstruction of network ecologies to rank all ecologies (i.e. nodes andedges) within each sample based on co-occurrence, [maximumco-occurrence; maximum co-occurrence less 1; maximum co-occurrence less2; etc.] until the networks with minimum co-occurrence are defined(i.e., a minimum edge score is achieved). This method can becomputationally intensive for data sets containing a large number ofsubjects. For data sets containing a large number of subjects thealgorithm uses a strategy whereby first seed network ecologies areconstructed using the method defined above in a subset of subjects andthen combinations of these seed networks are used to search for higherorder networks across the entire data set.

Biological significance can be assigned to the observed networkecologies and members of a given Network Ecology based on multiplecomputed metrics including, but not limited to: (i) the frequency that agiven OTU or Network Ecology is observed; (ii) the number of OTUs in thenetwork; the frequency of occurrence of the network across subjects(i.e., pervasiveness); (iii) the phylogenetic breadth of the network,(iv) specific functional properties, and (v) whether the network occurspreferentially in individuals that are healthy versus those harboringdisease (i.e., the various phenotypes). All network ecologies or OTUsthat occur in one phenotype (e.g., health) are compared to those thatoccur in other phenotypes (e.g., one or more specific disease states) tocore Network ecologies or OTUs that are found in one, two, or anymultiple of phenotypes. Network ecologies are considered to be relatedif at least 70%, 80%, or 90% of their OTUs are in common. All networkecologies or OTUs are assigned a score for their biological significancebased on but not limited to: (i) the intersections of phenotypes inwhich they occur or do not occur (e.g. present in health but notdisease), and (ii) the various metrics above defined. The final outputof all of these steps defines a set of Network Ecologies that are ofhigh biological significance and a set of Keystone OTUs and/or metabolicfunctions that are integral components of these derived ecologies.

From these Network Ecologies, the method includes defining “NetworkClasses” that represent network groups or clusters with specific,related compositional characteristics with respect to OTU content,phylogenetic diversity, and metabolic functional capacity (FIG. 18).Network Classes can be first defined by setting an inclusion thresholdfor networks to include in the analysis that is based on biologicalcharacteristics of the networks such as but not limited to the size(number of OTUs) and pervasiveness (i.e., how frequently a given networkis observed in a population of individuals). Selected network ecologiesare then clustered using two vectors: one vector is phylogeneticrelatedness of individual OTUs as defined by a computed phylogenetictree, and the second vector is network relatedness based on the OTUscontent in the individual networks. In another embodiment, clusteringvectors are related based on metabolic functional pathways harbored byindividual OTUs, and network relatedness is based on the functionalpathways present in each individual network. Network Classes are definedby specific nodes in the dendrogram representing the computed networkrelatedness, and each class is characterized by a specific combinationof OTUs. In one embodiment, these nodes are defined as branches of thehierarchical clustering tree based on the topological overlap measure;this measure is a highly robust measure of network interconnectedness.See Langfelder P, Zhang B, Horvath S. 2008. Defining clusters from ahierarchical cluster tree: the Dynamic Tree Cut package for R.Bioinformatics 24: 719-720.

From these Networks Classes, a target microbial composition'susefulness, e.g., as a therapeutic, is selected using desiredphylogenetic and functional properties for subsequent testing in invitro and in vivo models. Exemplary Network Classes are delineated inTable 12, and Table 13 defines taxonomic families that arecharacteristic of Network Classes.

As described herein, provided are compositions (Table 8) containingkeystone OTUs for states of health, including one or more of the OTUsprovided below in Table 9.

As described herein, provided are compositions containing keystone OTUs,keystone metabolic functions and, optionally, non-keystone OTUs,including one or more of the OTUs provided below in Table 10.

In some therapeutic compositions, keystone OTUs are provided frommembers of a genera or family selected from Table 9.

Exemplary network ecologies are provided in Table 8, Table 11, Table 12,Table 14a 14b, and 14c, and Table 17.

Exemlary functional network ecologies are provided in Table 18 and Table21.

Thus, provided herein are methods for production of a compositioncontaining a population of bacteria as either vegetative cells or sporesor both, suitable for therapeutic administration to a mammalian subjectin need thereof. The composition is produced by generally following thesteps of: (a) defining a target composition by selecting a NetworkEcology, Functional Network Ecology, a Network Class, or a set ofKeystone OTUs or Keystone Metabolic Functions that comprise the NetworkEcology or Functional Network Ecology of interest (a) providingbacterial OTUs obtained from one or more bacterial cultures, biologicalor environmental sources, or a mammalian donor subject; and (b)combining the bacterial OTUs in a ratio and an amount sufficient to forma Network Ecology or Functional Network Ecology. The composition isformulated such that a single oral dose contains at least about 1×10⁴colony forming units of the bacteria, and a single oral dose willtypically contain about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵CFUs of the bacteria. The concentration of bacterial of a given speciesor strain, or the aggregate of all species or strains, is e.g., 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ viable bacteria per gram ofcomposition or per administered dose.

The bacterial compositions are generally formulated for oral or gastricadministration, typically to a mammalian subject. In particularembodiments, the composition is formulated for oral administration as asolid, semi-solid, gel, or liquid form, such as in the form of a pill,tablet, capsule, or lozenge. In some embodiments, such formulationscontain or are coated by an enteric coating to protect the bacteriathrough the stomach and small intestine, although compositionscontaining spores are generally resistant to the environment of thestomach and small intestine. Alternatively, the bacterial compositionmay be formulated for naso-gastric or rectal administration.

The bacterial compositions may be formulated to be effective in a givenmammalian subject in a single administration or over multipleadministrations. For example, a single administration is substantiallyeffective to reduce Clostridium difficile (i.e., C. difficile) and/or C.difficile toxin content and/or toxin activity, in a mammalian subject towhom the composition is administered. Substantially effective means thatCl. difficile and/or C. difficile toxin content in the subject isreduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,98%, 99% or greater than 99% following administration of thecomposition.

Methods for Determining 16S Sequences

OTUs can be defined either by full 16S sequencing of the rRNA gene, bysequencing of a specific hypervariable region of this gene (i.e. V1, V2,V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination ofhypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial16S rDNA is approximately 1500 nucleotides in length and is used inreconstructing the evolutionary relationships and sequence similarity ofone bacterial isolate to another using phylogenetic approaches. 16Ssequences are used for phylogenetic reconstruction as they are ingeneral highly conserved, but contain specific hypervariable regionsthat harbor sufficient nucleotide diversity to differentiate genera andspecies of most microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S sequence, genomicDNA is extracted from a bacterial sample, the 16S rDNA (full region orspecific hypervariable regions) amplified using polymerase chainreaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingcan be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

OTUs can be defined by a combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof, full-genome sequence, or partial genome sequencegenerated using amplified genetic products, or whole genome sequence(WGS). Using well defined methods DNA extracted from a bacterial samplewill have specific genomic regions amplified using PCR and sequenced todetermine the nucleotide sequence of the amplified products. In thewhole genome shotgun (WGS) method, extracted DNA will be directlysequenced without amplification. Sequence data can be generated usingany sequencing technology including, but not limited to Sanger,Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences,and/or Oxford Nanopore.

Methods for Preparing a Bacterial Composition for Administration to aSubject

Methods for producing bacterial compositions can include three mainprocessing steps, combined with one or more mixing steps. The stepsinclude organism banking, organism production, and preservation.

For banking, the strains included in the bacterial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments that use a culturing step, the agar or broth can containnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. A variety of microbiological media and variations arewell known in the art (e.g. R. M. Atlas, Handbook of MicrobiologicalMedia (2010) CRC Press). Medium can be added to the culture at thestart, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of thebacterial composition, or as an entire collection comprising thebacterial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For bacterial compositions for humanuse, this is often at 37° C. temperature, pH, and other parameter withvalues similar to the normal human niche. The environment can beactively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions(e.g., gut microbiota), an anoxic/reducing environment can be employed.This can be accomplished by addition of reducing agents such as cysteineto the broth, and/or stripping it of oxygen. As an example, a culture ofa bacterial composition can be grown at 37° C., pH 7, in the mediumabove, pre-reduced with 1 g/L cysteine □HCl.

When the culture has generated sufficient biomass, it can be preservedfor banking. The organisms can be placed into a chemical milieu thatprotects from freezing (adding ‘cryoprotectants’), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termbacterial composition storage stability at temperatures elevated abovecryogenic. If the bacterial composition comprises spore forming speciesand results in the production of spores, the final composition can bepurified by additional means, such as density gradient centrifugationpreserved using the techniques described above. Bacterial compositionbanking can be done by culturing and preserving the strainsindividually, or by mixing the strains together to create a combinedbank. As an example of cryopreservation, a bacterial composition culturecan be harvested by centrifugation to pellet the cells from the culturemedium, the supernate decanted and replaced with fresh culture brothcontaining 15% glycerol. The culture can then be aliquoted into 1 mLcryotubes, sealed, and placed at −80° C. for long-term viabilityretention. This procedure achieves acceptable viability upon recoveryfrom frozen storage.

Organism production can be conducted using similar culture steps tobanking, including medium composition and culture conditions. It can beconducted at larger scales of operation, especially for clinicaldevelopment or commercial production. At larger scales, there can beseveral subcultivations of the bacterial composition prior to the finalcultivation. At the end of cultivation, the culture is harvested toenable further formulation into a dosage form for administration. Thiscan involve concentration, removal of undesirable medium components,and/or introduction into a chemical milieu that preserves the bacterialcomposition and renders it acceptable for administration via the chosenroute. For example, a bacterial composition can be cultivated to aconcentration of 10¹⁰ CFU/mL, then concentrated 20-fold by tangentialflow microfiltration; the spent medium can be exchanged by diafilteringwith a preservative medium consisting of 2% gelatin, 100 mM trehalose,and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder can be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

Methods of Treating a Subject

In some embodiments, the compositions disclosed herein are administeredto a patient or a user (sometimes collectively referred to as a“subject”). As used herein “administer” and “administration” encompassesembodiments in which one person directs another to consume a bacterialcomposition in a certain manner and/or for a certain purpose, and alsosituations in which a user uses a bacteria composition in a certainmanner and/or for a certain purpose independently of or in variance toany instructions received from a second person. Non-limiting examples ofembodiments in which one person directs another to consume a bacterialcomposition in a certain manner and/or for a certain purpose includewhen a physician prescribes a course of conduct and/or treatment to apatient, when a parent commands a minor user (such as a child) toconsume a bacterial composition, when a trainer advises a user (such asan athlete) to follow a particular course of conduct and/or treatment,and when a manufacturer, distributer, or marketer recommends conditionsof use to an end user, for example through advertisements or labeling onpackaging or on other materials provided in association with the sale ormarketing of a product.

The bacterial compositions offer a protective and/or therapeutic effectagainst diseases, disorders or conditions associated with dysbiosis ofthe gut microbiota, including but not limited to metabolic disorderssuch as pre-diabetes, type 1 diabetes, type 2 diabetes, obesity andnon-alcoholic fatty liver disease (NAFLD), gastrointestinal disorderssuch as inflammatory bowel disease (IBD, such as ulcerative colitis andCrohns' disease), pouchitis and irritable bowel syndrome (IBS), andinfectious diseases as described herein.

In some embodiments, the bacterial compositions offer a protectiveand/or therapeutic effect against diseases, disorders or conditionsassociated with dysbiosis of the gut microbiota, including but notlimited to, metabolic diseases (e.g., Type 1 diabetes, Type 2 diabetes,Gestational diabetes, Diabetes complications, Prediabetes, NAFLD/NASH,Obesity, Weight Loss), GI diseases (Inflammatory bowel disease (IBD),Irritable bowel syndrome (IBS), Ulcerative Colitis, Crohn's Disease).Infectious diseases (Clostridium difficile Associated Diarrhea (CDAD),Carbapenem-resistant Enterobacteriaceae (CRE), Multidrug-resistantAcinetobacter, Drug-resistant Campylobacter, Extended spectrumβ-lactamase producing Enterobacteriaceae (ESBLs), Vancomycin-resistantEnterococcus (VRE), Multidrug-resistant Pseudomonas aeruginosa,Drug-resistant Non-typhoidal Salmonella, Drug-resistant SalmonellaTyphi, Drug-resistant Shigella, Methicillin-resistant Staphylococcusaureus (MRSA), Drug-resistant Streptococcus pneumonia,Vancomycin-resistant Staphylococcus aureus (VRSA),Erythromycin-resistant Group A Streptococcus, Clindamycin-resistantGroup B Streptococcus, Pathogenic fungus, or Candida infection).

The present bacterial compositions can be administered to animals,including humans, laboratory animals (e.g., primates, rats, mice),livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), andhousehold pets (e.g., dogs, cats, rodents).

In the present method, the bacterial composition can be administeredenterically, in other words, by a route of access to thegastrointestinal tract. This includes oral administration, rectaladministration (including enema, suppository, or colonoscopy), by anoral or nasal tube (nasogastric, nasojejunal, oral gastric, or oraljejunal), as detailed more fully herein.

Pretreatment Protocols

Prior to administration of the bacterial composition, the patient canoptionally have a pretreatment protocol to prepare the gastrointestinaltract to receive the bacterial composition.

As one way of preparing the patient for administration of the microbialecosystem, at least one antibiotic can be administered to alter thebacteria in the patient. As another way of preparing the patient foradministration of the microbial ecosystem, a standard colon-cleansingpreparation can be administered to the patient to substantially emptythe contents of the colon, such as used to prepare a patient for acolonscopy. By “substantially emptying the contents of the colon,” thisapplication means removing at least 75%, at least 80%, at least 90%, atleast 95%, or about 100% of the contents of the ordinary volume of coloncontents. Antibiotic treatment can precede the colon-cleansing protocol.

If a patient has received an antibiotic for treatment of an infection,or if a patient has received an antibiotic as part of a specificpretreatment protocol, in one embodiment, the antibiotic can be stoppedin sufficient time to allow the antibiotic to be substantially reducedin concentration in the gut before the bacterial composition isadministered. In one embodiment, the antibiotic can be discontinued 1,2, or 3 days before the administration of the bacterial composition. Inanother embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7antibiotic half-lives before administration of the bacterialcomposition. In another embodiment, the antibiotic can be chosen so theconstituents in the bacterial composition have an MIC50 that is higherthan the concentration of the antibiotic in the gut.

MIC50 of a bacterial composition or the elements in the composition canbe determined by methods well known in the art. Reller et al.,Antimicrobial Susceptibility Testing: A Review of General Principles andContemporary Practices, Clinical Infectious Diseases 49(11):1749-1755(2009). In such an embodiment, the additional time between antibioticadministration and administration of the bacterial composition is notnecessary. If the pretreatment protocol is part of treatment of an acuteinfection, the antibiotic can be chosen so that the infection issensitive to the antibiotic, but the constituents in the bacterialcomposition are not sensitive to the antibiotic.

Administration of Bacterial Compositions

The bacterial compositions of the invention are suitable foradministration to mammals and non-mammalian animals in need thereof. Incertain embodiments, the mammalian subject is a human subject who hasone or more symptoms of a dysbiosis.

When the mammalian subject is suffering from a disease, disorder orcondition characterized by an aberrant microbiota, the bacterialcompositions described herein are suitable for treatment thereof. Insome embodiments, the mammalian subject has not received antibiotics inadvance of treatment with the bacterial compositions. For example, themammalian subject has not been administered at least two doses ofvancomycin, metronidazole and/or or similar antibiotic compound withinone week prior to administration of the therapeutic composition. Inother embodiments, the mammalian subject has not previously received anantibiotic compound in the one month prior to administration of thetherapeutic composition. In other embodiments, the mammalian subject hasreceived one or more treatments with one or more different antibioticcompounds and such treatment(s) resulted in no improvement or aworsening of symptoms.

In some embodiments, the gastrointestinal disease, disorder or conditionis diarrhea caused by C. difficile including recurrent C. difficileinfection, ulcerative colitis, colitis, Crohn's disease, or irritablebowel disease. Beneficially, the therapeutic composition is administeredonly once prior to improvement of the disease, disorder or condition. Insome embodiments the therapeutic composition is administered atintervals greater than two days, such as once every three, four, five orsix days, or every week or less frequently than every week. Or thepreparation may be administered intermittently according to a setschedule, e.g., once a day, once weekly, or once monthly, or when thesubject relapses from the primary illness. In another embodiment, thepreparation may be administered on a long-term basis to subjects who areat risk for infection with or who may be carriers of these pathogens,including subjects who will have an invasive medical procedure (such assurgery), who will be hospitalized, who live in a long-term care orrehabilitation facility, who are exposed to pathogens by virtue of theirprofession (livestock and animal processing workers), or who could becarriers of pathogens (including hospital workers such as physicians,nurses, and other health care professionals).

In embodiments, the bacterial composition is administered enterically.This preferentially includes oral administration, or by an oral or nasaltube (including nasogastric, nasojejunal, oral gastric, or oraljejunal). In other embodiments, administration includes rectaladministration (including enema, suppository, or colonoscopy). Thebacterial composition may be administered to at least one region of thegastrointestinal tract, including the mouth, esophagus, stomach, smallintestine, large intestine, and rectum. In some embodiments it isadministered to all regions of the gastrointestinal tract. The bacterialcompositions may be administered orally in the form of medicaments suchas powders, capsules, tablets, gels or liquids. The bacterialcompositions may also be administered in gel or liquid form by the oralroute or through a nasogastric tube, or by the rectal route in a gel orliquid form, by enema or instillation through a colonoscope or by asuppository.

If the composition is administered colonoscopically and, optionally, ifthe bacterial composition is administered by other rectal routes (suchas an enema or suppository) or even if the subject has an oraladministration, the subject may have a colon cleansing preparation. Thecolon-cleansing preparation can facilitate proper use of the colonoscopeor other administration devices, but even when it does not serve amechanical purpose it can also maximize the proportion of the bacterialcomposition relative to the other organisms previously residing in thegastrointestinal tract of the subject. Any ordinarily acceptable coloncleansing preparation may be used such as those typically provided whena subject undergoes a colonoscopy.

Dosages and Schedule for Administration

In some embodiments, the bacteria and bacterial compositions areprovided in a dosage form. In certain embodiments, the dosage form isdesigned for administration of at least one OTU or combination thereofdisclosed herein, wherein the total amount of bacterial compositionadministered is selected from 0.1 ng to 10 g, 10 ng to 1 g, 100 ng to0.1 g, 0.1 mg to 500 mg, 1 mg to 100 mg, or from 10-15 mg. In otherembodiments, the bacterial composition is consumed at a rate of from 0.1ng to 10 g a day, 10 ng to 1 g a day, 100 ng to 0.1 g a day, 0.1 mg to500 mg a day, 1 mg to 100 mg a day, or from 10-15 mg a day, or more.

In certain embodiments, the treatment period is at least 1 day, at least2 days, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, or at least 1 year. Insome embodiments the treatment period is from 1 day to 1 week, from 1week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months,from 6 months to 1 year, or for over a year.

In one embodiment, 10⁵ and 10¹² microorganisms total can be administeredto the patient in a given dosage form. In another embodiment, aneffective amount can be provided in from 1 to 500 ml or from 1 to 500grams of the bacterial composition having from 10⁷ to 10¹¹ bacteria perml or per gram, or a capsule, tablet or suppository having from 1 mg to1000 mg lyophilized powder having from 10⁷ to 10¹¹ bacteria. Thosereceiving acute treatment can receive higher doses than those who arereceiving chronic administration.

Any of the preparations described herein can be administered once on asingle occasion or on multiple occasions, such as once a day for severaldays or more than once a day on the day of administration (includingtwice daily, three times daily, or up to five times daily). In anotherembodiment, the preparation can be administered intermittently accordingto a set schedule, e.g., once weekly, once monthly, or when the patientrelapses from the primary illness. In one embodiment, the preparationcan be administered on a long-term basis to individuals who are at riskfor infection with or who may be carriers of these pathogens.

Patient Selection

Particular bacterial compositions can be selected for individualpatients or for patients with particular profiles. For example, 16Ssequencing can be performed for a given patient to identify the bacteriapresent in his or her microbiota. The sequencing can either profile thepatient's entire microbiome using 16S sequencing (to the family, genera,or species level), a portion of the patient's microbiome using 16Ssequencing, or it can be used to detect the presence or absence ofspecific candidate bacteria that are biomarkers for health or aparticular disease state. Based on the biomarker data, a particularcomposition can be selected for administration to a patient tosupplement or complement a patient's microbiota in order to restorehealth or treat or prevent disease. In another embodiment, patients canbe screened to determine the composition of their microbiota todetermine the likelihood of successful treatment.

Combination Therapy

The bacterial compositions can be administered with other agents in acombination therapy mode, including anti-microbial agents andprebiotics. Administration can be sequential, over a period of hours ordays, or simultaneous.

In one embodiment, the bacterial compositions are included incombination therapy with one or more anti-microbial agents, whichinclude anti-bacterial agents, anti-fungal agents, anti-viral agents andanti-parasitic agents.

Anti-bacterial agents can include cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

Anti-viral agents can include Abacavir, Acyclovir, Adefovir, Amprenavir,Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol,Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine,Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine,Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Nevirapine,Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine,Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine,Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir,Zalcitabine, Zanamivir and Zidovudine.

Examples of antifungal compounds include, but are not limited to polyeneantifungals such as natamycin, rimocidin, filipin, nystatin,amphotericin B, candicin, and hamycin; imidazole antifungals such asmiconazole, ketoconazole, clotrimazole, econazole, omoconazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, and tioconazole; triazole antifungals suchas fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole,voriconazole, terconazole, and albaconazole; thiazole antifungals suchas abafungin; allylamine antifungals such as terbinafine, naftifine, andbutenafine; and echinocandin antifungals such as anidulafungin,caspofungin, and micafungin. Other compounds that have antifungalproperties include, but are not limited to polygodial, benzoic acid,ciclopirox, tolnaftate, undecylenic acid, flucytosine or5-fluorocytosine, griseofulvin, and haloprogin.

In one embodiment, the bacterial compositions are included incombination therapy with one or more corticosteroids, mesalazine,mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressivedrugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone,methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines, and combinations thereof.

A prebiotic is a selectively fermented ingredient that allows specificchanges, both in the composition and/or activity in the gastrointestinalmicrobiota that confers benefits upon host well-being and health.Prebiotics can include complex carbohydrates, amino acids, peptides, orother essential nutritional components for the survival of the bacterialcomposition. Prebiotics include, but are not limited to, amino acids,biotin, fructooligosaccharide, galactooligosaccharides, inulin,lactulose, mannan oligosaccharides, oligofructose-enriched inulin,oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide,and xylooligosaccharides.

Methods for Testing Bacterial Compositions for Populating Effect

In Vivo Assay for Determining Whether a Bacterial Composition Populatesa Subject's Gastrointestinal Tract

In order to determine that the bacterial composition populates thegastrointestinal tract of a subject, an animal model, such as a mousemodel, can be used. The model can begin by evaluating the microbiota ofthe mice. Qualitative assessments can be accomplished using 16Sprofiling of the microbial community in the feces of normal mice. It canalso be accomplished by full genome sequencing, whole genome shotgunsequencing (WGS), or traditional microbiological techniques.Quantitative assessments can be conducted using quantitative PCR (qPCR),described below, or by using traditional microbiological techniques andcounting colony formation.

Optionally, the mice can receive an antibiotic treatment to mimic thecondition of dysbiosis. Antibiotic treatment can decrease the taxonomicrichness, diversity, and evenness of the community, including areduction of abundance of a significant number of bacterial taxa.Dethlefsen et al., The pervasive effects of an antibiotic on the humangut microbiota, as revealed by deep 16S rRNA sequencing, PLoS Biology6(11):3280 (2008). At least one antibiotic can be used, and antibioticsare well known. Antibiotics can include aminoglycoside antibiotic(amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin, andapramycin), amoxicillin, ampicillin, Augmentin (anamoxicillin/clavulanate potassium combination), cephalosporin (cefaclor,defadroxil, cefazolin, cefixime, fefoxitin, cefprozil, ceftazimdime,cefuroxime, cephalexin), clavulanate potassium, clindamycin, colistin,gentamycin, kanamycin, metronidazole, or vancomycin. As an individual,nonlimiting specific example, the mice can be provided with drinkingwater containing a mixture of the antibiotics kanamycin, colistin,gentamycin, metronidazole and vancomycin at 40 mg/kg, 4.2 mg/kg, 3.5mg/kg, 21.5 mg/kg, and 4.5 mg/kg (mg per average mouse body weight),respectively, for 7 days. Alternatively, mice can be administeredciprofloxacin at a dose of 15-20 mg/kg (mg per average mouse bodyweight), for 7 days. If the mice are provided with an antibiotic, a washout period of from one day to three days may be provided with noantibiotic treatment and no bacterial composition treatment.

Subsequently, the bacterial composition is administered to the mice byoral gavage. The bacterial composition may be administered in a volumeof 0.2 ml containing 10⁴ CFUs of each type of bacteria in the bacterialcomposition. Dose-response may be assessed by using a range of doses,including, but not limited to 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,and/or 10¹⁰.

The mice can be evaluated using 16S sequencing, full genome sequencing,whole genome shotgun sequencing (WGS), or traditional microbiologicaltechniques to determine whether the bacterial composition has populatedthe gastrointestinal tract of the mice. For example only, one day, threedays, one week, two weeks, and one month after administration of thebacterial composition to the mice, 16S profiling is conducted todetermine whether the test bacterial composition has populated thegastrointestinal tract of the mice. Quantitative assessments, includingqPCR and traditional microbiological techniques such as colony counting,can additionally or alternatively be performed, at the same timeintervals.

Furthermore, the number of sequence counts that correspond exactly tothose in the bacterial composition over time can be assessed todetermine specifically which components of the bacterial compositionreside in the gastrointestinal tract over a particular period of time.In one embodiment, the strains of the bacterial composition persist fora desired period of time. In another embodiment, the components of thebacterial composition persist for a desired period of time, while alsoincreasing the ability of other microbes (such as those present in theenvironment, food, etc.) to populate the gastrointestinal tract, furtherincreasing overall diversity, as discussed below.

Ability of Bacterial compositions to Populate Different Regions of theGastrointestinal Tract

The present bacterial compositions can also be assessed for theirability to populate different regions of the gastrointestinal tract. Inone embodiment, a bacterial composition can be chosen for its ability topopulate one or more than one region of the gastrointestinal tract,including, but not limited to the stomach, the small intestine(duodenum, jejunum, and ileum), the large intestine (the cecum, thecolon (the ascending, transverse, descending, and sigmoid colon), andthe rectum).

An in vivo study can be conducted to determine which regions of thegastrointestinal tract a given bacterial composition will populate. Amouse model similar to the one described above can be conducted, exceptinstead of assessing the feces produced by the mice, particular regionsof the gastrointestinal tract can be removed and studied individually.For example, at least one particular region of the gastrointestinaltract can be removed and a qualitative or quantitative determination canbe performed on the contents of that region of the gastrointestinaltract. In another embodiment, the contents can optionally be removed andthe qualitative or quantitative determination may be conducted on thetissue removed from the mouse.

qPCR

As one quantitative method for determining whether a bacterialcomposition populates the gastrointestinal tract, quantitative PCR(qPCR) can be performed. Standard techniques can be followed to generatea standard curve for the bacterial composition of interest, either forall of the components of the bacterial composition collectively,individually, or in subsets (if applicable). Genomic DNA can beextracted from samples using commercially-available kits, such as the MoBio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

In some embodiments, qPCR can be conducted using HotMasterMix (5PRIME,Gaithersburg, Md.) and primers specific for the bacterial composition ofinterest, and may be conducted on a MicroAmpx Fast Optical 96-wellReaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island,N.Y.) and performed on a BioRad C1000™ Thermal Cycler equipped with aCFX96™ Real-Time System (BioRad, Hercules, Calif.), with fluorescentreadings of the FAM and ROX channels. The Cq value for each well on theFAM channel is determined by the CFX Manager™ software version 2.1. Thelog₁₀(cfu/ml) of each experimental sample is calculated by inputting agiven sample's Cq value into linear regression model generated from thestandard curve comparing the Cq values of the standard curve wells tothe known log₁₀(cfu/ml) of those samples. The skilled artisan may employalternative qPCR modes.

Methods for Characterization of Bacterial Compositions

In certain embodiments, provided are methods for testing certaincharacteristics of bacterial compositions. For example, the sensitivityof bacterial compositions to certain environmental variables isdetermined, e.g., in order to select for particular desirablecharacteristics in a given composition, formulation and/or use. Forexample, the constituents in the bacterial composition can be tested forpH resistance, bile acid resistance, and/or antibiotic sensitivity,either individually on a constituent-by-constituent basis orcollectively as a bacterial composition comprised of multiple bacterialconstituents (collectively referred to in this section as bacterialcomposition).

pH Sensitivity Testing

If a bacterial composition will be administered other than to the colonor rectum (i.e., for example, an oral route), optionally testing for pHresistance enhances the selection of bacterial compositions that willsurvive at the highest yield possible through the varying pHenvironments of the distinct regions of the GI tract. Understanding howthe bacterial compositions react to the pH of the GI tract also assistsin formulation, so that the number of bacteria in a dosage form can beincreased if beneficial and/or so that the composition may beadministered in an enteric-coated capsule or tablet or with a bufferingor protective composition. As the pH of the stomach can drop to a pH of1 to 2 after a high-protein meal for a short time before physiologicalmechanisms adjust it to a pH of 3 to 4 and often resides at a resting pHof 4 to 5, and as the pH of the small intestine can range from a pH of 6to 7.4, bacterial compositions can be prepared that survive thesevarying pH ranges (specifically wherein at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% of thebacteria can survive gut transit times through various pH ranges). Thiscan be tested by exposing the bacterial composition to varying pH rangesfor the expected gut transit times through those pH ranges. Therefore,as a nonlimiting example only, 18-hour cultures of bacterialcompositions can be grown in standard media, such as gut microbiotamedium (“GMM”, see Goodman et al., Extensive personal human gutmicrobiota culture collections characterized and manipulated ingnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or anotheranimal-products-free medium, with the addition of pH adjusting agentsfor a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1 hour, a pH of 4to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3 hours. Analternative method for testing stability to acid is described in U.S.Pat. No. 4,839,281. Survival of bacteria may be determined by culturingthe bacteria and counting colonies on appropriate selective ornon-selective media.

Bile Acid Sensitivity Testing

Additionally, in some embodiments, testing for bile-acid resistanceenhances the selection of bacterial compositions that will surviveexposures to bile acid during transit through the GI tract. Bile acidsare secreted into the small intestine and can, like pH, affect thesurvival of bacterial compositions. This can be tested by exposing thebacterial compositions to bile acids for the expected gut exposure timeto bile acids. For example, bile acid solutions can be prepared atdesired concentrations using 0.05 mM Tris at pH 9 as the solvent. Afterthe bile acid is dissolved, the pH of the solution may be adjusted to7.2 with 10% HCl. Bacterial compositions can be cultured in 2.2 ml of abile acid composition mimicking the concentration and type of bile acidsin the patient, 1.0 ml of 10% sterile-filtered feces media and 0.1 ml ofan 18-hour culture of the given strain of bacteria. Incubations may beconducted for from 2.5 to 3 hours or longer. An alternative method fortesting stability to bile acid is described in U.S. Pat. No. 4,839,281.Survival of bacteria can be determined by culturing the bacteria andcounting colonies on appropriate selective or non-selective media.

Antibiotic Sensitivity Testing

As a further optional sensitivity test, bacterial compositions can betested for sensitivity to antibiotics. In one embodiment, bacterialcompositions can be chosen so that the bacterial constituents aresensitive to antibiotics such that if necessary they can be eliminatedor substantially reduced from the patient's gastrointestinal tract by atleast one antibiotic targeting the bacterial composition.

Adherence to Gastrointestinal Cells

The bacterial compositions may optionally be tested for the ability toadhere to gastrointestinal cells. A method for testing adherence togastrointestinal cells is described in U.S. Pat. No. 4,839,281.

Methods for Purifying Spores

Solvent Treatments

To purify the bacterial spores, the fecal material is subjected to oneor more solvent treatments. A solvent treatment is a miscible solventtreatment (either partially miscible or fully miscible) or an immisciblesolvent treatment. Miscibility is the ability of two liquids to mix witheach to form a homogeneous solution. Water and ethanol, for example, arefully miscible such that a mixture containing water and ethanol in anyratio will show only one phase. Miscibility is provided as a wt/wt %, orweight of one solvent in 100 g of final solution. If two solvents arefully miscible in all proportions, their miscibility is 100%. Providedas fully miscible solutions with water are alcohols, e.g., methanol,ethanol, isopropanol, butanol, etc. The alcohols can be provided alreadycombined with water; e.g., a solution containing 10%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% orgreater than 95% Other solvents are only partially miscible, meaningthat only some portion will dissolve in water. Diethyl ether, forexample, is partially miscible with water. Up to 7 grams of diethylether will dissolve in 93 g of water to give a 7% (wt/wt %) solution. Ifmore diethyl ether is added, a two phase solution will result with adistinct diethyl ether layer above the water. Other miscible materialsinclude ethers, dimethoxyethane, or tetrahydrofuran In contrast, an oilsuch as an alkane and water are immiscible and form two phases. Further,immiscible treatments are optionally combined with a detergent, eitheran ionic detergent or a non-ionic detergent. Exemplary detergentsinclude Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or apolyol.

Chromatography Treatments

To purify spore populations, the fecal materials are subjected to one ormore chromatographic treatments, either sequentially or in parallel. Ina chromatographic treatment, a solution containing the fecal material iscontacted with a solid medium containing a hydrophobic interactionchromatographic (HIC) medium or an affinity chromatographic medium. Inan alternative embodiment, a solid medium capable of absorbing aresidual habitat product present in the fecal material is contacted witha solid medium that adsorbs a residual habitat product. In certainembodiments, the HIC medium contains sepharose or a derivatizedsepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, orbutyl-s sepharose. In other embodiments, the affinity chromatographicmedium contains material derivatized with mucin type I, II, III, IV, V,or VI, or oligosaccharides derived from or similar to those of mucinstype I, II, III, IV, V, or VI. Alternatively, the affinitychromatographic medium contains material derivatized with antibodiesthat recognize spore-forming bacteria.

Mechanical Treatments

Provided herein is the physical disruption of the fecal material,particularly by one or more mechanical treatment such as blending,mixing, shaking, vortexing, impact pulverization, and sonication. Asprovided herein, the mechanical disrupting treatment substantiallydisrupts a non-spore material present in the fecal material and does notsubstantially disrupt a spore present in the fecal material. Mechanicaltreatments optionally include filtration treatments, where the desiredspore populations are retained on a filter while the undesirable(non-spore) fecal components to pass through, and the spore fraction isthen recovered from the filter medium. Alternatively, undesirableparticulates and eukaryotic cells may be retained on a filter whilebacterial cells including spores pass through. In some embodiments thespore fraction retained on the filter medium is subjected to adiafiltration step, wherein the retained spores are contacted with awash liquid, typically a sterile saline-containing solution or otherdiluent, in order to further reduce or remove the undesirable fecalcomponents.

Thermal Treatments

Provided herein is the thermal disruption of the fecal material.Generally, the fecal material is mixed in a saline-containing solutionsuch as phosphate-buffered saline (PBS) and subjected to a heatedenvironment, such as a warm room, incubator, water-bath, or the like,such that efficient heat transfer occurs between the heated environmentand the fecal material. Preferably the fecal material solution is mixedduring the incubation to enhance thermal conductivity and disruptparticulate aggregates. Thermal treatments can be modulated by thetemperature of the environment and/or the duration of the thermaltreatment. For example, the fecal material or a liquid comprising thefecal material is subjected to a heated environment, e.g., a hot waterbath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at leastabout 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more than 10 hours. In certain embodiments the thermal treatmentoccurs at two different temperatures, such as 30 seconds in a 100 degreeCelsius environment followed by 10 minutes in a 50 degree Celsiusenvironment. In preferred embodiments the temperature and duration ofthe thermal treatment are sufficient to kill or remove pathogenicmaterials while not substantially damaging or reducing thegermination-competency of the spores.

Irradiation Treatments

Provided are methods of treating the fecal material or separatedcontents of the fecal material with ionizing radiation, typically gammairradiation, ultraviolet irradiation or electron beam irradiationprovided at an energy level sufficient to kill pathogenic materialswhile not substantially damaging the desired spore populations. Forexample, ultraviolet radiation at 254 nm provided at an energy levelbelow about 22,000 microwatt seconds per cm² will not generally destroydesired spores.

Centrifugation and Density Separation Treatments

Provided are methods of separating desired spore populations from theother components of the fecal material by centrifugation. A solutioncontaining the fecal material is subjected to one or more centrifugationtreatments, e.g., at about 1000×g, 2000×g, 3000×g, 4000×g, 5000×g,6000×g, 7000×g, 8000×g or greater than 8000×g. Differentialcentrifugation separates desired spores from undesired non-sporematerial; at low forces the spores are retained in solution, while athigher forces the spores are pelleted while smaller impurities (e.g.,virus particles, phage) are retained in solution. For example, a firstlow force centrifugation pellets fibrous materials; a second, higherforce centrifugation pellets undesired eukaryotic cells, and a third,still higher force centrifugation pellets the desired spores while smallcontaminants remain in suspension. In some embodiments density ormobility gradients or cushions (e.g., step cushions), such as Percoll,Ficoll, Nycodenz, Histodenz or sucrose gradients, are used to separatedesired spore populations from other materials in the fecal material.

Also provided herein are methods of producing spore populations thatcombine two or more of the treatments described herein in order tosynergistically purify the desired spores while killing or removingundesired materials and/or activities from the spore population. It isgenerally desirable to retain the spore populations undernon-germinating and non-growth promoting conditions and media, in orderto minimize the growth of pathogenic bacteria present in the sporepopulations and to minimize the germination of spores into vegetativebacterial cells.

Pharmaceutical Compositions and Formulations of the Invention

Formulations

Provided are formulations for administration to humans and othersubjects in need thereof. Generally the bacterial compositions arecombined with additional active and/or inactive materials in order toproduce a final product, which may be in single dosage unit or in amulti-dose format.

In some embodiments, the composition comprises at least onecarbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars.The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate can be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates can contain modified saccharideunits, such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates can exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. Asused herein, a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments, the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In other embodiments, thecomposition comprises at least one modified lipid, for example, a lipidthat has been modified by cooking.

In some embodiments, the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In certain embodiments, the composition comprises at least onesupplemental vitamin. The at least one vitamin can be fat-soluble orwater soluble vitamins. Suitable vitamins include but are not limited tovitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin,niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine,pantothenic acid, and biotin. Suitable forms of any of the foregoing aresalts of the vitamin, derivatives of the vitamin, compounds having thesame or similar activity of the vitamin, and metabolites of the vitamin.

In other embodiments, the composition comprises an excipient.Non-limiting examples of suitable excipients include a buffering agent,a preservative, a stabilizer, a binder, a compaction agent, a lubricant,a dispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In another embodiment, the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In other embodiments, the composition comprises a binder as anexcipient. Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In another embodiment, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In other embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In other embodiments, the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-crystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pecitin, andtragacanth. In another embodiment, the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In another embodiment, the excipient comprises a flavoring agent.Flavoring agents can be chosen from synthetic flavor oils and flavoringaromatics; natural oils; extracts from plants, leaves, flowers, andfruits; and combinations thereof. In some embodiments the flavoringagent is selected from cinnamon oils; oil of wintergreen; peppermintoils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oilsuch as lemon oil, orange oil, grape and grapefruit oil; and fruitessences including apple, peach, pear, strawberry, raspberry, cherry,plum, pineapple, and apricot.

In other embodiments, the excipient comprises a sweetener. Non-limitingexamples of suitable sweeteners include glucose (corn syrup), dextrose,invert sugar, fructose, and mixtures thereof (when not used as acarrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; and sugar alcohols such as sorbitol,mannitol, sylitol, and the like. Also contemplated are hydrogenatedstarch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In yet other embodiments, the composition comprises a coloring agent.Non-limiting examples of suitable color agents include food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), and externaldrug and cosmetic colors (Ext. D&C). The coloring agents can be used asdyes or their corresponding lakes.

The weight fraction of the excipient or combination of excipients in theformulation is usually about 99% or less, such as about 95% or less,about 90% or less, about 85% or less, about 80% or less, about 75% orless, about 70% or less, about 65% or less, about 60% or less, about 55%or less, 50% or less, about 45% or less, about 40% or less, about 35% orless, about 30% or less, about 25% or less, about 20% or less, about 15%or less, about 10% or less, about 5% or less, about 2% or less, or about1% or less of the total weight of the composition.

The bacterial compositions disclosed herein can be formulated into avariety of forms and administered by a number of different means. Thecompositions can be administered orally, rectally, or parenterally, informulations containing conventionally acceptable carriers, adjuvants,and vehicles as desired. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, or intrasternal injection andinfusion techniques. In an exemplary embodiment, the bacterialcomposition is administered orally.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, troches, lozenges, powders, and granules. A capsuletypically comprises a core material comprising a bacterial compositionand a shell wall that encapsulates the core material. In someembodiments, the core material comprises at least one of a solid, aliquid, and an emulsion. In other embodiments, the shell wall materialcomprises at least one of a soft gelatin, a hard gelatin, and a polymer.Suitable polymers include, but are not limited to: cellulosic polymerssuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose (HPMC), methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulosesuccinate and carboxymethylcellulose sodium; acrylic acid polymers andcopolymers, such as those formed from acrylic acid, methacrylic acid,methyl acrylate, ammonio methylacrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate (e.g., those copolymers soldunder the trade name “Eudragit”); vinyl polymers and copolymers such aspolyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate,vinylacetate crotonic acid copolymer, and ethylene-vinyl acetatecopolymers; and shellac (purified lac). In yet other embodiments, atleast one polymer functions as taste-masking agents.

Tablets, pills, and the like can be compressed, multiply compressed,multiply layered, and/or coated. The coating can be single or multiple.In one embodiment, the coating material comprises at least one of asaccharide, a polysaccharide, and glycoproteins extracted from at leastone of a plant, a fungus, and a microbe. Non-limiting examples includecorn starch, wheat starch, potato starch, tapioca starch, cellulose,hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin,mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gumkaraya, gum ghatti, tragacanth gum, funori, carrageenans, agar,alginates, chitosans, or gellan gum. In some embodiments the coatingmaterial comprises a protein. In another embodiment, the coatingmaterial comprises at least one of a fat and an oil. In otherembodiments, the at least one of a fat and an oil is high temperaturemelting. In yet another embodiment, the at least one of a fat and an oilis hydrogenated or partially hydrogenated. In one embodiment, the atleast one of a fat and an oil is derived from a plant. In otherembodiments, the at least one of a fat and an oil comprises at least oneof glycerides, free fatty acids, and fatty acid esters. In someembodiments, the coating material comprises at least one edible wax. Theedible wax can be derived from animals, insects, or plants. Non-limitingexamples include beeswax, lanolin, bayberry wax, carnauba wax, and ricebran wax. Tablets and pills can additionally be prepared with entericcoatings.

Alternatively, powders or granules embodying the bacterial compositionsdisclosed herein can be incorporated into a food product. In someembodiments, the food product is a drink for oral administration.Non-limiting examples of a suitable drink include fruit juice, a fruitdrink, an artificially flavored drink, an artificially sweetened drink,a carbonated beverage, a sports drink, a liquid diary product, a shake,an alcoholic beverage, a caffeinated beverage, infant formula and soforth. Other suitable means for oral administration include aqueous andnonaqueous solutions, emulsions, suspensions and solutions and/orsuspensions reconstituted from non-effervescent granules, containing atleast one of suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, coloring agents, and flavoringagents.

In some embodiments, the food product can be a solid foodstuff. Suitableexamples of a solid foodstuff include without limitation a food bar, asnack bar, a cookie, a brownie, a muffin, a cracker, an ice cream bar, afrozen yogurt bar, and the like.

In other embodiments, the compositions disclosed herein are incorporatedinto a therapeutic food. In some embodiments, the therapeutic food is aready-to-use food that optionally contains some or all essentialmacronutrients and micronutrients. In another embodiment, thecompositions disclosed herein are incorporated into a supplementary foodthat is designed to be blended into an existing meal. In one embodiment,the supplemental food contains some or all essential macronutrients andmicronutrients. In another embodiment, the bacterial compositionsdisclosed herein are blended with or added to an existing food tofortify the food's protein nutrition. Examples include food staples(grain, salt, sugar, cooking oil, margarine), beverages (coffee, tea,soda, beer, liquor, sports drinks), snacks, sweets and other foods.

In one embodiment, the formulations are filled into gelatin capsules fororal administration. An example of an appropriate capsule is a 250 mggelatin capsule containing from 10 (up to 100 mg) of lyophilized powder(10⁸ to 10¹¹ bacteria), 160 mg microcrystalline cellulose, 77.5 mggelatin, and 2.5 mg magnesium stearate. In an alternative embodiment,from 10⁵ to 10¹² bacteria may be used, 10⁵ to 10⁷, 10⁶ to 10⁷, or 10⁸ to10¹⁰, with attendant adjustments of the excipients if necessary. In analternative embodiment, an enteric-coated capsule or tablet or with abuffering or protective composition can be used.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B(1992).

Example 1: Sequence-based Genomic Characterization of OperationalTaxonomic Units (OTU) and Functional Genes

Method for Determining 16S rDNA Gene Sequence

As described above, OTUs are defined either by full 16S sequencing ofthe rRNA gene, by sequencing of a specific hypervariable region of thisgene (i.e. V1, V2, V3, V4, V5, V6, V7, V8, or V9), or by sequencing ofany combination of hypervariable regions from this gene (e.g. V1-3 orV3-5). The bacterial 16S rRNA gene is approximately 1500 nucleotides inlength and is used in reconstructing the evolutionary relationships andsequence similarity of one bacterial isolate to another usingphylogenetic approaches. 16S sequences are used for phylogeneticreconstruction as they are in general highly conserved, but containspecific hypervariable regions that harbor sufficient nucleotidediversity to differentiate genera and species of most microbes. rRNAgene sequencing methods are applicable to both the analysis ofnon-enriched samples, but also for identification of microbes afterenrichment steps that either enrich the microbes of interest from themicrobial composition and/or the nucleic acids that harbor theappropriate rDNA gene sequences as described below. For example,enrichment treatments prior to 16S rDNA gene characterization willincrease the sensitivity of 16S as well as other molecular-basedcharacterization nucleic acid purified from the microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S rRNA sequence,genomic DNA is extracted from a bacterial sample, the 16S rDNA (fullregion or specific hypervariable regions) amplified using polymerasechain reaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingmay be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

Method for Determining 18S rDNA and ITS Gene Sequence

Methods to assign and identify fungal OTUs by genetic means can beaccomplished by analyzing 18S sequences and the internal transcribedspacer (ITS). The rRNA of fungi that forms the core of the ribosome istranscribed as a signal gene and consists of the 8S, 5.8S and 28Sregions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28Sregions, respectively. These two intercistronic segments between the 18Sand 5.8S and 5.8S and 28S regions are removed by splicing and containsignificant variation between species for barcoding purposes aspreviously described (Schoch et al Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker forFungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used forphylogenetic reconstruction however the ITS can serve this function asit is generally highly conserved but contains hypervariable regions thatharbor sufficient nucleotide diversity to differentiate genera andspecies of most fungus.

Using well known techniques, in order to determine the full 18S and ITSsequences or a smaller hypervariable section of these sequences, genomicDNA is extracted from a microbial sample, the rDNA amplified usingpolymerase chain reaction (PCR), the PCR products cleaned, andnucleotide sequences delineated to determine the genetic compositionrDNA gene or subdomain of the gene. The sequencing method used may be,but is not limited to, Sanger sequencing or using a next-generationsequencing method, such as an Illumina (sequencing by synthesis) methodusing barcoded primers allowing for multiplex reactions.

Method for Determining Other Marker Gene Sequences

In addition to the 16S and 18S rRNA gene, one may define an OTU bysequencing a selected set of genes that are known to be marker genes fora given species or taxonomic group of OTUs. These genes mayalternatively be assayed using a PCR-based screening strategy. Asexample, various strains of pathogenic Escherichia coli can bedistinguished using DNAs from the genes that encode heat-labile (LTI,LTIIa, and LTIIb) and heat-stable (STI and STII) toxins, verotoxin types1, 2, and 2e (VT1, VT2, and VT2e, respectively), cytotoxic necrotizingfactors (CNF1 and CNF2), attaching and effacing mechanisms (eaeA),enteroaggregative mechanisms (Eagg), and enteroinvasive mechanisms(Einv). The optimal genes to utilize for taxonomic assignment of OTUs byuse of marker genes will be familiar to one with ordinary skill of theart of sequence based taxonomic identification.

Genomic DNA Extraction

Genomic DNA is extracted from pure microbial cultures using a hotalkaline lysis method. 1 μl of microbial culture is added to 9 μl ofLysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. andneutralized by the addition of 10 μl of Neutralization Buffer (40 mMTris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).Alternatively, genomic DNA is extracted from pure microbial culturesusing commercially available kits such as the Mo Bio Ultraclean®Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) orby standard methods known to those skilled in the art. For fungalsamples, DNA extraction can be performed by methods described previously(US20120135127) for producing lysates from fungal fruiting bodies bymechanical grinding methods.

Amplification of 16S Sequences for Downstream Sanger Sequencing

To amplify bacterial 16S rDNA (e.g, in FIG. 1), 2 μl of extracted gDNAis added to a 20 μl final volume PCR reaction. For full-length 16sequencing the PCR reaction also contains 1× HotMasterMix (5PRIME,Gaithersburg, Md.), 250 nM of 27f (AGRGTTTGATCMTGGCTCAG (SEQ ID NO:2033), IDT, Coralville, Iowa), and 250 nM of 1492r(TACGGYTACCTTGTTAYGACTT (SEQ ID NO: 2034), IDT, Coralville, Iowa), withPCR Water (Mo Bio Laboratories, Carlsbad, Calif.) for the balance of thevolume.

FIG. 1 shows the hypervariable regions mapped onto a 16s sequence andthe sequence regions corresponding to these sequences on a sequence map.A schematic is shown of a 16S rRNA gene and the figure denotes thecoordinates of hypervariable regions 1-9 (V1-V9), according to anembodiment of the invention. Coordinates of V1-V9 are 69-99, 137-242,433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294, and 1435-1465respectively, based on numbering using E. coli system of nomenclaturedefined by Brosius et al., Complete nucleotide sequence of a 16Sribosomal RNA gene (16S rRNA) from Escherichia coli, PNAS75(10):4801-4805 (1978).

Alternatively, other universal bacterial primers or thermostablepolymerases known to those skilled in the art are used. For example,primers are available to those skilled in the art for the sequencing ofthe the “V1-V9 regions” of the 16S rRNA (e.g., FIG. 1). These regionsrefer to the first through ninth hypervariable regions of the 16S rRNAgene that are used for genetic typing of bacterial samples. Theseregions in bacteria are defined by nucleotides 69-99, 137-242, 433-497,576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465respectively using numbering based on the E. coli system ofnomenclature. See Brosius et al., Complete nucleotide sequence of a 16Sribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978).In some embodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8,and V9 regions are used to characterize an OTU. In one embodiment, theV1, V2, and V3 regions are used to characterize an OTU. In anotherembodiment, the V3, V4, and V5 regions are used to characterize an OTU.In another embodiment, the V4 region is used to characterize an OTU. Aperson of ordinary skill in the art can identify the specifichypervariable regions of a candidate 16S rRNA (e.g., FIG. 1) bycomparing the candidate sequence in question to the reference sequence(as in FIG. 2) and identifying the hypervariable regions based onsimilarity to the reference hypervariable regions. FIG. 2 highlights inbold the nucleotide sequences for each hypervariable region in theexemplary reference E. coli 16S sequence described by Brosius et al.

The PCR is performed on commercially available thermocyclers such as aBioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). Thereactions are run at 94° C. for 2 minutes followed by 30 cycles of 94°C. for 30 seconds, 51° C. for 30 seconds, and 68° C. for 1 minute 30seconds, followed by a 7 minute extension at 72° C. and an indefinitehold at 4° C. Following PCR, gel electrophoresis of a portion of thereaction products is used to confirm successful amplification of a ˜1.5kb product.

To remove nucleotides and oligonucleotides from the PCR products, 2 μlof HT ExoSap-IT (Affymetrix, Santa Clara, Calif.) is added to 5 μl ofPCR product followed by a 15 minute incubation at 37° C. and then a 15minute inactivation at 80° C.

Amplification of 16S Sequences for Downstream Characterization ByMassively Parallel Sequencing Technologies

Amplification performed for downstream sequencing by short readtechnologies such as Illumina require amplification using primers knownto those skilled in the art that additionally include a sequence-basedbarcoded tag. As example, to amplify the 16s hypervariable region V4region of bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20 μlfinal volume PCR reaction. The PCR reaction also contains 1×HotMasterMix (5PRIME, Gaithersburg, Md.), 200 nM of V4_515f_adapt(AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCCGCGG TAA (SEQ IDNO: 2035), IDT, Coralville, Iowa), and 200 nM of barcoded 806rbc(CAAGCAGAAGACGGCATACGAGAT_12bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NOs: 2036-2037, respectively, in order ofappearance), IDT, Coralville, Iowa), with PCR Water (Mo BioLaboratories, Carlsbad, Calif.) for the balance of the volume. Theseprimers incorporate barcoded adapters for Illumina sequencing bysynthesis. Optionally, identical replicate, triplicate, or quadruplicatereactions may be performed. Alternatively other universal bacterialprimers or thermostable polymerases known to those skilled in the artare used to obtain different amplification and sequencing error rates aswell as results on alternative sequencing technologies.

The PCR amplification is performed on commercially availablethermocyclers such as a BioRad MyCycler™ Thermal Cycler (BioRad,Hercules, Calif.). The reactions are run at 94° C. for 3 minutesfollowed by 25 cycles of 94° C. for 45 seconds, 50° C. for 1 minute, and72° C. for 1 minute 30 seconds, followed by a 10 minute extension at 72°C. and a indefinite hold at 4° C. Following PCR, gel electrophoresis ofa portion of the reaction products is used to confirm successfulamplification of a ˜1.5 kb product. PCR cleanup is performed asdescribed above.

Sanger Sequencing of Target Amplicons from Pure Homogeneous Samples

To detect nucleic acids for each sample, two sequencing reactions areperformed to generate a forward and reverse sequencing read. Forfull-length 16s sequencing primers 27f and 1492r are used. 40 ng ofExoSap-IT-cleaned PCR products are mixed with 25 pmol of sequencingprimer and Mo Bio Molecular Biology Grade Water (Mo Bio Laboratories,Carlsbad, Calif.) to 15 μl total volume. This reaction is submitted to acommercial sequencing organization such as Genewiz (South Plainfield,N.J.) for Sanger sequencing.

Amplification of 18S and ITS regions for Downstream Sequencing

To amplify the 18S or ITS regions, 2 μL fungal DNA were amplified in afinal volume of 30 μL with 15 μL AmpliTaq Gold 360 Mastermix, PCRprimers, and water. The forward and reverse primers for PCR of the ITSregion are 5′-TCCTCCGCTTATTGATATGC-3′ (SEQ ID NO: 2038) and5′-GGAAGTAAAAGTCGTAACAAGG-3′ (SEQ ID NO: 2039) and are added at 0.2 uMconcentration each. The forward and reverse primers for the 18s regionare 5′-GTAGTCATATGCTTGTCTC-3′ (SEQ ID NO: 2040) and5′-CTTCCGTCAATTCCTTTAAG-3′ (SEQ ID NO: 2041) and are added at 0.4 uMconcentration each. PCR is performed with the following protocol: 95 Cfor 10 min, 35 cycles of 95 C for 15 seconds, 52 C for 30 seconds, 72 Cfor 1.5 s; and finally 72 C for 7 minutes followed by storage at 4 C.All forward primers contained the M13F-20 sequencing primer, and reverseprimers included the M13R-27 sequencing primer. PCR products (3 μL) wereenzymatically cleaned before cycle sequencing with 1 μL ExoSap-IT and 1μL Tris EDTA and incubated at 37° C. for 20 min followed by 80° C. for15 min. Cycle sequencing reactions contained 5 μL cleaned PCR product, 2μL BigDye Terminator v3.1 Ready Reaction Mix, 1 μL 5× Sequencing Buffer,1.6 pmol of appropriate sequencing primers designed by one skilled inthe art, and water in a final volume of 10 μL. The standard cyclesequencing protocol is 27 cycles of 10 s at 96° C., 5 s at 50° C., 4 minat 60° C., and hold at 4° C. Sequencing cleaning is performed with theBigDye XTerminator Purification Kit as recommended by the manufacturerfor 10-μL volumes. The genetic sequence of the resulting 18S and ITSsequences is performed using methods familiar to one with ordinary skillin the art using either Sanger sequencing technology or next-generationsequencing technologies such as but not limited to Illumina.

Preparation of Extracted Nucleic Acids for Metagenomic Characterizationby Massively Parallel Sequencing Technologies

Extracted nucleic acids (DNA or RNA) are purified and prepared bydownstream sequencing using standard methods familiar to one withordinary skill in the art and as described by the sequencingtechnology's manufactures instructions for library preparation. Inshort, RNA or DNA are purified using standard purification kits such asbut not limited to Qiagen's RNeasy Kit or Promega's Genomic DNApurification kit. For RNA, the RNA is converted to cDNA prior tosequence library construction. Following purification of nucleic acids,RNA is converted to cDNA using reverse transcription technology such asbut not limited to Nugen Ovation RNA-Seq System or Illumina Truseq asper the manufacturer's instructions. Extracted DNA or transcribed cDNAare sheared using physical (e.g., Hydroshear), acoustic (e.g., Covaris),or molecular (e.g., Nextera) technologies and then size selected as perthe sequencing technologies manufacturer's recommendations. Followingsize selection, nucleic acids are prepared for sequencing as per themanufacturer's instructions for sample indexing and sequencing adapterligation using methods familiar to one with ordinary skill in the art ofgenomic sequencing.

Massively Parallel Sequencing of Target Amplicons from HeterogeneousSamples DNA Quantification & Library Construction

The cleaned PCR amplification products are quantified using theQuant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island,N.Y.) according to the manufacturer's instructions. Followingquantification, the barcoded cleaned PCR products are combined such thateach distinct PCR product is at an equimolar ratio to create a preparedIllumina library.

Nucleic Acid Detection

The prepared library is sequenced on Illumina HiSeq or MiSeq sequencers(Illumina, San Diego, Calif.) with cluster generation, templatehybridization, isothermal amplification, linearization, blocking anddenaturation and hybridization of the sequencing primers performedaccording to the manufacturer's instructions. 16SV4SeqFw(TATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 2042)), 16SV4SeqRev(AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO. 2037)), and 16SV4Index(ATTAGAWACCCBDGTAGTCCGGCTGACTGACT (SEQ ID NO: 2043) (IDT, Coralville,Iowa) are used for sequencing. Other sequencing technologies can be usedsuch as but not limited to 454, Pacific Biosciences, Helicos, IonTorrent, and Nanopore using protocols that are standard to someoneskilled in the art of genomic sequencing.

Example 2. Sequence Read Annotation

Primary Read Annotation

Nucleic acid sequences are analyzed and annotated to define taxonomicassignments using sequence similarity and phylogenetic placement methodsor a combination of the two strategies. A similar approach can be usedto annotate protein names, protein function, transcription factor names,and any other classification schema for nucleic acid sequences. Sequencesimilarity based methods include those familiar to individuals skilledin the art including, but not limited to BLAST, BLASTx, tBLASTn,tBLASTx, RDP-classifier, DNAclust, and various implementations of thesealgorithms such as Qiime or Mothur. These methods rely on mapping asequence read to a reference database and selecting the match with thebest score and e-value. Common databases include, but are not limited tothe Human Microbiome Project, NCBI non-redundant database, Greengenes,RDP, and Silva for taxonomic assignments. For functional assignmentsreads are mapped to various functional databases such as but not limitedto COG, KEGG, BioCyc, and MetaCyc. Further functional annotations can bederived from 16S taxonomic annotations using programs such as PICRUST(M. Langille, et al 2013. Nature Biotechnology 31, 814-821).Phylogenetic methods can be used in combination with sequence similaritymethods to improve the calling accuracy of an annotation or taxonomicassignment. Here tree topologies and nodal structure are used to refinethe resolution of the analysis. In this approach we analyze nucleic acidsequences using one of numerous sequence similarity approaches andleverage phylogenetic methods that are well known to those skilled inthe art, including but not limited to maximum likelihood phylogeneticreconstruction (see e.g. Liu K, Linder C R, and Warnow T. 2011. RAxMLand FastTree: Comparing Two Methods for Large-Scale Maximum LikelihoodPhylogeny Estimation. PLoS ONE 6: e27731. McGuire G, Denham M C, andBalding D J. 2001. Models of sequence evolution for DNA sequencescontaining gaps. Mol. Biol. Evol 18: 481-490. Wróbel B. 2008.Statistical measures of uncertainty for branches in phylogenetic treesinferred from molecular sequences by using model-based methods. J. Appl.Genet. 49: 49-67.) Sequence reads (e.g. 16S, 18S, or ITS) are placedinto a reference phylogeny comprised of appropriate reference sequences.Annotations are made based on the placement of the read in thephylogenetic tree. The certainty or significance of the OTU annotationis defined based on the OTU's sequence similarity to a reference nucleicacid sequence and the proximity of the OTU sequence relative to one ormore reference sequences in the phylogeny. As an example, thespecificity of a taxonomic assignment is defined with confidence at thethe level of Family, Genus, Species, or Strain with the confidencedetermined based on the position of bootstrap supported branches in thereference phylogenetic tree relative to the placement of the OTUsequence being interrogated. Nucleic acid sequences can be assignedfunctional annotations using the methods described above.

Clade Assignments

The ability of 16S-V4 OTU identification to assign an OTU as a specificspecies depends in part on the resolving power of the 16S-V4 region ofthe 16S gene for a particular species or group of species. Both thedensity of available reference 16S sequences for different regions ofthe tree as well as the inherent variability in the 16S gene betweendifferent species will determine the definitiveness of a taxonomicannotation. Given the topological nature of a phylogenetic tree and thefact that tree represents hierarchical relationships of OTUs to oneanother based on their sequence similarity and an underlyingevolutionary model, taxonomic annotations of a read can be rolled up toa higher level using a clade-based assignment procedure. Using thisapproach, clades are defined based on the topology of a phylogenetictree that is constructed from full-length 16S sequences using maximumlikelihood or other phylogenetic models familiar to individuals withordinary skill in the art of phylogenetics. Clades are constructed toensure that all OTUs in a given clade are: (i) within a specified numberof bootstrap supported nodes from one another (generally, 1-5bootstraps), and (ii) share a defined percent similarity (for 16Smolecular data typically set to 95%-97% sequence similarity). OTUs thatare within the same clade can be distinguished as genetically andphylogenetically distinct from OTUs in a different clade based on 16S-V4sequence data. OTUs falling within the same clade are evolutionarilyclosely related and may or may not be distinguishable from one anotherusing 16S-V4 sequence data. The power of clade based analysis is thatmembers of the same clade, due to their evolutionary relatedness, arelikely to play similar functional roles in a microbial ecology such asthat found in the human gut. Compositions substituting one species withanother from the same clade are likely to have conserved ecologicalfunction and therefore are useful in the present invention. Notably inaddition to 16S-V4 sequences, clade-based analysis can be used toanalyze 18S, ITS, and other genetic sequences.

Notably, 16S sequences of isolates of a given OTU are phylogeneticallyplaced within their respective clades, sometimes in conflict with themicrobiological-based assignment of species and genus that may havepreceded 16S-based assignment. Discrepancies between taxonomicassignment based on microbiological characteristics versus geneticsequencing are known to exist from the literature.

For a given network ecology or functional network ecology one can definea set of OTUs from the network's representative clades. As example, if anetwork was comprised of clade_100 and clade_102 it can be said to becomprised of at least one OTU from the group consisting ofCorynebacterium coyleae, Corynebacterium mucifaciens, andCorynebacterium ureicelerivorans, and at least one OTU from the groupconsisting of Corynebacterium appendicis, Corynebacterium genitalium,Corynebacterium glaucum, Corynebacterium imitans, Corynebacteriumriegelii, Corynebacterium sp. L_2012475, Corynebacterium sp. NML93_0481, Corynebacterium sundsvallense, and Corynebacterium tuscaniae(see Table 1). Conversely as example, if a network was said to consistof Corynebacterium coyleae and/or Corynebacterium mucifaciens and/orCorynebacterium ureicelerivorans, and also consisted of Corynebacteriumappendicis and/or Corynebacterium genitalium and/or Corynebacteriumglaucum and/or Corynebacterium imitans and/or Corynebacterium riegeliiand/or Corynebacterium sp. L_2012475 and/or Corynebacterium sp. NML93_0481 and/or Corynebacterium sundsvallense and/or Corynebacteriumtuscaniae it can be said to be comprised of clade_100 and clade_102.

The applicants made clade assignments to all OTUs reported in theapplication using the above described method and these assignments arereported in Table 1. In some embodiments, the network analysis permitssubstitutions of clade_172 by clade_172i. In another embodiment, thenetwork analysis permits substitutions of clade_198 by clade_198i. Inanother embodiment, the network analysis permits substitutions ofclade_260 by clade_260c, clade_260g or clade_260h. In anotherembodiment, the network analysis permits substitutions of clade_262 byclade_262i. In another embodiment, the network analysis permitssubstitutions of clade_309 by clade_309c, clade_309e, clade_309g,clade_309h or clade_309i. In another embodiment, the network analysispermits substitutions of clade_313 by clade_313f. In another embodiment,the network analysis permits substitutions of clade_325 by clade_325f.In another embodiment, the network analysis permits substitutions ofclade_335 by clade_335i. In another embodiment, the network analysispermits substitutions of clade_351 by clade_351e. In another embodiment,the network analysis permits substitutions of clade_354 by clade_354e.In another embodiment, the network analysis permits substitutions ofclade_360 by clade_360c, clade_360g, clade_360h, or clade_360i. Inanother embodiment, the network analysis permits substitutions ofclade_378 by clade_378e. In another embodiment, the network analysispermits substitutions of clade_38 by clade_38e or clade_38i. In anotherembodiment, the network analysis permits substitutions of clade_408 byclade_408b, clade_408d, clade_408f, clade_408g or clade_408h. In anotherembodiment, the network analysis permits substitutions of clade_420 byclade_420f. In another embodiment, the network analysis permitssubstitutions of clade_444 by clade_444i. In another embodiment, thenetwork analysis permits substitutions of clade_478 by clade_478i. Inanother embodiment, the network analysis permits substitutions ofclade_479 by clade_479c, by clade_479g or by clade_479h. In anotherembodiment, the network analysis permits substitutions of clade_481 byclade_481a, clade_481b, clade_481e, clade_481g, clade_481h or byclade_481i. In another embodiment, the network analysis permits thenetwork analysis permits substitutions of clade_497 by clade_497e or byclade_497f. In another embodiment, the network analysis permits thenetwork analysis permits substitutions of clade_512 by clade_512i. Inanother embodiment, the network analysis permits the network analysispermits substitutions of clade_516 by clade_516c, by clade_516g or byclade_516h. In another embodiment, the network analysis permits thenetwork analysis permits substitutions of clade_522 by clade_522i. Inanother embodiment, the network analysis permits the network analysispermits substitutions of clade_553 by clade_553i. In another embodiment,the network analysis permits the network analysis permits substitutionsof clade_566 by clade_566f. In another embodiment, the network analysispermits the network analysis permits substitutions of clade_572 byclade_572i. In another embodiment, the network analysis permits thenetwork analysis permits substitutions of clade_65 by clade_65e. Inanother embodiment, the network analysis permits the network analysispermits substitutions of clade_92 by clade_92e or by clade_92i. Inanother embodiment, the network analysis permits the network analysispermits substitutions of clade_96 by clade_96g or by clade_96h. Inanother embodiment, the network analysis permits the network analysispermits substitutions of clade_98 by clade_98i. These permitted cladesubstitutions are described in Table 22.

Metagenomic Read Annotation

Metagenomic or whole genome shotgun sequence data is annotated asdescribed above, with the additional step that sequences are eitherclustered or assembled prior to annotation. Following sequencecharacterization as described above, sequence reads are demultiplexedusing the indexing (i.e. barcodes). Following demultiplexing sequencereads are either: (i) clustered using a rapid clustering algorithm suchas but not limited to UCLUST (http://drive5.com/usearch/manual/uclustalgo.html) or hash methods such VICUNA (Xiao Yang, Patrick Charlebois,Sante Gnerre, Matthew G Coole, Niall J. Lennon, Joshua Z. Levin, JamesQu, Elizabeth M. Ryan, Michael C. Zody, and Matthew R. Henn. 2012. Denovo assembly of highly diverse viral populations. BMC Genomics 13:475).Following clustering a representative read for each cluster isidentified based and analyzed as described above in “Primary ReadAnnotation”. The result of the primary annotation is then applied to allreads in a given cluster. (ii) A second strategy for metagenomicsequence analysis is genome assembly followed by annotation of genomicassemblies using a platform such as but not limited to MetAMOS (T J.Treangen et al. 2013 Geneome Biology 14:R2), HUMAaN (Abubucker S, SegataN, Goll J, Schubert A M, Izard J, Cantarel B L, Rodriguez-Mueller B,Zucker J, Thiagaraj an M, Henrissat B, et al. 2012. MetabolicReconstruction for Metagenomic Data and Its Application to the HumanMicrobiome ed. J. A. Eisen. PLoS Computational Biology 8: e1002358) andother methods familiar to one with ordinary skill in the art.

Example 3. OTU Identification Using Microbial Culturing Techniques

The identity of the bacterial species which grow up from a complexfraction can be determined in multiple ways. First, individual coloniesare picked into liquid media in a 96 well format, grown up and saved as15% glycerol stocks at −80° C. Aliquots of the cultures are placed intocell lysis buffer and colony PCR methods can be used to amplify andsequence the 16S rDNA gene (Example 1). Alternatively, colonies arestreaked to purity in several passages on solid media. Well separatedcolonies are streaked onto the fresh plates of the same kind andincubated for 48-72 hours at 37° C. The process is repeated multipletimes in order to ensure purity. Pure cultures are analyzed byphenotypic- or sequence-based methods, including 16S rDNA amplificationand sequencing as described in Example 1. Sequence characterization ofpure isolates or mixed communities e.g. plate scrapes and sporefractions can also include whole genome shotgun sequencing. The latteris valuable to determine the presence of genes associated withsporulation, antibiotic resistance, pathogenicity, and virulence.Colonies are also scraped from plates en masse and sequenced using amassively parallel sequencing method as described in Example 1, suchthat individual 16S signatures can be identified in a complex mixture.Optionally, the sample can be sequenced prior to germination (ifappropriate DNA isolation procedures are used to lsye and release theDNA from spores) in order to compare the diversity of germinable specieswith the total number of species in a spore sample. As an alternative orcomplementary approach to 16S analysis, MALDI-TOF-mass spec is used forspecies identification (Barreau M, Pagnier I, La Scola B. 2013.Improving the identification of anaerobes in the clinical microbiologylaboratory through MALDI-TOF mass spectrometry. Anaerobe 22: 123-125).

Example 4. Microbiological Strain Identification Approaches

Pure bacterial isolates are identified using microbiological methods asdescribed in Wadsworth-KTL Anaerobic Microbiology Manual(Jouseimies-Somer H, Summanen P H, Citron D, Baron E, Wexler H M,Finegold S M. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual), andThe Manual of Clinical Microbiology (ASM Press, 10th Edition). Thesemethods rely on phenotypes of strains and include Gram-staining toconfirm Gram positive or negative staining behavior of the cellenvelope, observance of colony morphologies on solid media, motility,cell morphology observed microscopically at 60× or 100× magnificationincluding the presence of bacterial endospores and flagella. Biochemicaltests that discriminate between genera and species are performed usingappropriate selective and differential agars and/or commerciallyavailable kits for identification of Gram negative and Gram positivebacteria and yeast, for example, RapID tests (Remel) or API tests(bioMerieux). Similar identification tests can also be performed usinginstrumentation such as the Vitek 2 system (bioMerieux). Phenotypictests that discriminate between genera and species and strains (forexample the ability to use various carbon and nitrogen sources) can alsobe performed using growth and metabolic activity detection methods, forexample the Biolog Microbial identification microplates. The profile ofshort chain fatty acid production during fermentation of particularcarbon sources can also be used as a way to discriminate between species(Wadsworth-KTL Anaerobic Microbiology Manual, Jousimies-Somer, et al2002). MALDI-TOF-mass spectrometry can also be used for speciesidentification (as reviewed in Anaerobe 22:123).

Example 5. Computational Prediction of Network Ecologies

Source data comprising a genomic-based characterization of a microbiomeof individual samples were used as input computationally delineatenetwork ecologies that would have biological properties that arecharacteristic of a state of health and could catalzye a shift from astate of microbial dysbiosis to a state of health. Applicants obtained16S and metagenomic sequence datasets from public data repositories (seee.g. The Human Microbiome Project Consortium. 2012. Structure, functionand diversity of the healthy human microbiome. Nature 486: 207-214. Dataaccessible at URL: hmpdacc.org) and MetaHit Project (Arumugam M, Raes J,Pelletier E, Paslier D L, Yamada T, Mende D R, Fernandes G R, Tap J,Bruls T, Batto J-M, et al. 2011. Enterotypes of the human gutmicrobiome. Nature 473: 174-180. Data accessible at URL: metahit.eu) forrelevant microbiome studies in multiple disease indications includingCDAD, Type 2 Diabetes, Ulcerative Colitis, and Irritable Bowel Disease,or generated data sets from samples directly using the methods describedin Examples 1 & 2 and further described in the literature (see e.g.Aagaard K, Riehle K, Ma J, Segata N, Mistretta T-A, Coarfa C, Raza S,Rosenbaum S, Van den Veyver I, Milosavljevic A, et al. 2012. AMetagenomic Approach to Characterization of the Vaginal MicrobiomeSignature in Pregnancy ed. A. J. Ratner. PLoS ONE 7: e36466. JumpstartConsortium Human Microbiome Project Data Generation Working Group. 2012.Evaluation of 16S rDNA-Based Community Profiling for Human MicrobiomeResearch ed. J. Ravel. PLoS ONE 7: e39315. The Human Microbiome ProjectConsortium. 2012. Structure, function and diversity of the healthy humanmicrobiome. Nature 486: 207-214.). Nucleic acid sequences were analyzedand taxonomic and phylogenetic assignments of specific OTUs were madeusing sequence similarity and phylogenetic methods that are well knownto those skilled in the art, including but not limited to maximumlikelihood phylogenetic reconstruction (see e.g. Liu K, Linder C R, andWarnow T. 2011. RAxML and FastTree: Comparing Two Methods forLarge-Scale Maximum Likelihood Phylogeny Estimation. PLoS ONE 6: e27731.McGuire G, Denham M C, and Balding D J. 2001. Models of sequenceevolution for DNA sequences containing gaps. Mol. Biol. Evol 18:481-490. Wróbel B. 2008. Statistical measures of uncertainty forbranches in phylogenetic trees inferred from molecular sequences byusing model-based methods. J. Appl. Genet. 49: 49-67.) From thesetaxonomic assignments OTUs and clades in the dataset were defined usingthe method described in Examples 1 and 2. The certainty of the OTU callwas defined based on the OTU's sequence similarity to a referencenucleic acid sequence and the proximity of the OTU sequence relative toone or more reference sequences in the phylogeny. The specificity of anOTU's taxonomic and phlylogenetic assignment determines whether thematch is assigned at the level of Family, Genus, Species, or Strain, andthe confidence of this assignment is determined based on the position ofbootstrap supported branches in the reference phylogenetic tree relativeto the placement of the OTU sequence being interrogated. In addition,microbial OTU assignments may be obtained from assignments made inpeer-reviewed publications.

Applicants designated individual subject samples to biologicallyrelevant sample phenotypes such as but not limited to “healthy state,”“recurrent Clostridium difficile infection,” “Crohn's disease,” “InsulinResistance,” “Obesity,” “Type 2 diabetes,” “Ulcerative Colitis”. In oneembodiment samples are assigned to “health” and “disease” phenotypes. Inanother embodiment, samples are assigned higher resolution phenotypesuch as but not limited to: “health:human”, “health:mouse”,“health:human microbiome project”, “health:microbiota donor”,“health:microbiota recipient”, “disease:microbiota recipient”, or“disease:no treatment”, “disease:human”, or “disease:mouse”. In anotherembodiment, samples where assigned to higher resolution phenotypes, suchas but not limited to those defined in FIG. 19 that characterizephenotypes specific to samples from fecal donors and patients whoreceived a fecal microbial transplant from these donors. FIG. 19 showsphenotypes assigned to samples for the computational derivation ofNetwork Ecologies that typify microbiome states of health (Hpost, Hdon,& Hgen) and states of disease (DdonF & DpreF).

In another embodiment, other phenotypes that define a category ofdisease or health that represents the underlying state of the populationunder study can be used. Applicants then computationally determined themicrobial network ecologies for each phenotype using the OTU and cladeassignments that comprise the microbial profile for each sample and thealgorithms described above in the Section entitled “Method ofDetermining Network Ecologies.”

Tables 8, 11, and 14a below provide exemplary network ecologies thatdefine states of health as compared to states of dysbiosis or diseasefor multiple disease indications. The disease indications for which thenetwork ecologies represent a health state are denoted in Table 8, andKeystone and Non-Keystone OTUs (see Example 6) are delineated in Tables9-10. Importantly, Network Ecologies that represent a state of health inone disease indication can represent states of health in additionaldisease states. Additionally, Keystone OTUs found in a networkassociated with health for different disease indications can overlap.Applicants found that a large number of network ecologies overlappedparticularly between those associated with health in the cases of CDADand Type 2 Diabetes despite the analysis of substantially differentgenomic data sets for the two diseases.

Example 6. Identification of Network Classes, Keystone OTUs, Clades, andFunctional Modalities

Identification of Keystone OTUs, Clades and Functions

The human body is an ecosystem in which the microbiota and themicrobiome play a significant role in the basic healthy function ofhuman systems (e.g. metabolic, immunological, and neurological). Themicrobiota and resulting microbiome comprise an ecology ofmicroorganisms that co-exist within single subjects interacting with oneanother and their host (i.e., the mammalian subject) to form a dynamicunit with inherent biodiversity and functional characteristics. Withinthese networks of interacting microbes (i.e. ecologies), particularmembers can contribute more significantly than others; as such thesemembers are also found in many different ecologies, and the loss ofthese microbes from the ecology can have a significant impact on thefunctional capabilities of the specific ecology. Robert Paine coined theconcept “Keystone Species” in 1969 (see Paine R T. 1969. A note ontrophic complexity and community stability. The American Naturalist 103:91-93) to describe the existence of such lynchpin species that areintegral to a given ecosystem regardless of their abundance in theecological community. Paine originally describe the role of the starfishPisaster ochraceus in marine systems and since the concept has beenexperimentally validated in numerous ecosystems.

Keystone OTUs (as shown in Table 9), Phylogenetic Clades (a.k.a.Clades), and/or Functions (for example, but not limited to, KEGGOrthology Pathways) are computationally-derived by analysis of networkecologies elucidated from a defined set of samples that share a specificphenotype. Keystone OTUs, Clades and/or Functions are defined as allNodes within a defined set of networks that meet two or more of thefollowing criteria. Using Criterion 1, the node is frequently observedin networks, and the networks in which the node is observed are found ina large number of individual subjects; the frequency of occurrence ofthese Nodes in networks and the pervasiveness of the networks inindividuals indicates these Nodes perform an important biologicalfunction in many individuals. Using Criterion 2, the node is frequentlyobserved in networks, and the Node is observed contains a large numberof edges connecting it to other nodes in the network. These Nodes arethus “super-connectors”, meaning that they form a nucleus of a majorityof networks (See FIG. 17) and as such have high biological significancewith respect to their functional contributions to a given ecology.

FIG. 17 is a schematic representation of how Keystone OTUs (nodes 2 and4, shaded circles) are central members of many network ecologies thatcontain non-Keystone OTUs (nodes 1, 3, and 5-9). Distinct networkecologies include [node 2--node 7], [node--3--node 2--node--4], [node2--node 4--node 5--node 6--node 7], [node 1--node 2--node 8--node 9],and [node--node 3].

Using Criterion 3, the Node is found in networks containing a largenumber of Nodes (i.e., they are large networks), and the networks inwhich the Node is found occur in a large number of subjects; thesenetworks are potentially of high interest as it is unlikely that largenetworks occurring in many individuals would occur by chance alonestrongly suggesting biological relevance. Optionally, the requiredthresholds for the frequency at which a Node is observed in networkecologies, the frequency at which a given network is observed acrosssubject samples, and the size of a given network to be considered aKeystone Node are defined by the 50th, 70th, 80th, or 90th percentilesof the distribution of these variables. Optionally, the requiredthresholds are defined by the value for a given variable that issignificantly different from the mean or median value for a givenvariable using standard parametric or non-parametric measures ofstatistical significance. In another embodiment a Keystone Node isdefined as one that occurs in a sample phenotype of interest such as butnot limited to “health” and simultaneously does not occur in a samplephenotype that is not of interest such as but not limited to “disease.”Optionally, a Keystone Node is defined as one that is shown to besignificantly different from what is observed using permuted testdatasets to measure significance. In another embodiment of Criterion 2Keystone OTUs, Clades, or Functions can be defined using a hierarchicalclustering method that clusters Networks based on their OTU, Clade, orfunctional pathways. Statistically significant branch points in thehierarchy are defined based on the topological overlap measure; thismeasure is a highly robust measure of network interconnectedness(Langfelder P, Zhang B, Horvath S. 2008. Defining clusters from ahierarchical cluster tree: the Dynamic Tree Cut package for R.Bioinformatics 24: 719-720.). Once these branch points are defined theKeystones are delineated as OTUs, clades or functional pathways that arefound consistently across all networks in all or a subset of the networkclusters.

Applicants defined the Keystone OTUs and Clades characteristic of healthstates for the computationally determined networks reported in Table 8for the various disease indications analyszed using the three criteriondefined above. Keystone Clades were defined from the Keystone OTUs usingclade definitions as outlined in Example 1. Keystone OTUs are reportedin Table 9. Importantly, we identified the absence of Keystone OTUs inmultiple particular disease states, indicating that bacterialcompositions comprised of specific sets of Keystone OTUs are likely tohave utility in multiple disease indications.

Demonstration that Keystone OTUs inhibit C. difficile Growth in aCompetitive In Vitro Simulation Assay

To screen the ability of binary combinations comprising at least oneKeystone OTU (binary pairs) to inhibit the growth of Clostridiumdifficile in vitro, vials of −80° C. glycerol stock banks of each OTUwere thawed and diluted to 1e8 CFU/mL. Each strain was then diluted 10×(to a final concentration of 1e7 CFU/mL of each strain) into 200 uL ofPBS+15% glycerol in the wells of a 96-well plate. Plates were thenfrozen at −80° C. When needed for the assay, plates were removed from−80° C. and thawed at room temperature under anaerobic conditions priorto use.

An overnight culture of Clostridium difficile was grown under anaerobicconditions in SweetB-FosIn or other suitable media for the growth of C.difficile. SweetB-FosIn is a complex media composed of brain heartinfusion, yeast extract, cysteine, cellobiose, maltose, soluble starch,and fructooligosaccharides/inulin, and hemin, and is buffered withmorpholino-propane sulphonic acid (MOPS). After 24 hr of growth theculture was diluted 100,000 fold into SweetB-FosIn. The diluted C.difficile mixture was then aliquoted to wells of a 96-well plate (180 uLto each well). 20 uL of a unique binary pair of Keystone OTUs was thenadded to each well at a final concentration of 1e6 CFU/mL of eachspecies. Alternatively the assay can be tested with binary pairs atdifferent initial concentrations (1e9 CFU/mL, 1e8 CFU/mL, 1e7 CFU/mL,1e5 CFU/mL, 1e4 CFU/mL, 1e3 CFU/mL, 1e2 CFU/mL). Control wells onlyinoculated with C. difficile were included for a comparison to thegrowth of C. difficile without inhibition. Additional wells were usedfor controls that either inhibit or do not inhibit the growth of C.difficile. Plates were wrapped with parafilm and incubated for 24 hr at37° C. under anaerobic conditions. After 24 hr the wells containing C.difficile alone were serially diluted and plated to determine titer onselective media such as CCFA (Anaerobe Systems) or CDSA (BectonDickinson). The 96-well plate was then frozen at −80° C. beforequantifying C. difficile by qPCR.

C. difficile in each well was quantified by qPCR. A standard curve wasgenerated from a well on each assay plate containing only pathogenic C.difficile grown in SweetB+FosIn media as provided herein and compare tothe microbiological titer determined above. Genomic DNA was extractedfrom the standard curve samples along with the other wells. Genomic DNAwas extracted from 5 μl of each sample using a dilution, freeze/thaw,and heat lysis protocol. 5 μL of thawed samples were added to 45 μL ofUltraPure water (Life Technologies, Carlsbad, Calif.) and mixed bypipetting. The plates with diluted samples were frozen at −20° C. untiluse for qPCR which includes a heated lysis step prior to amplification.Alternatively the genomic DNA could be isolated using the Mo BioPowersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,Carlsbad, Calif.), Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

The qPCR reaction mixture contained 1× SsoAdvanced Universal ProbesSupermix, 900 nM of Wr-tcdB-F primer (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG(SEQ ID NO: 2044), IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R primer(CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO: 2045), IDT, Coralville, Iowa),250 nM of Wr-tcdB-P probe (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQID NO. 2046), Life Technologies, Grand Island, N.Y.), and MolecularBiology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl(Primers adapted from: Wroblewski, D. et al., Rapid MolecularCharacterization of Clostridium difficile and Assessment of Populationsof C. difficile in Stool Specimens, Journal of Clinical Microbiology47:2142-2148 (2009)). This reaction mixture was aliquoted to wells of aHard-shell Low-Profile Thin Wall 96-well Skirted PCR Plate (BioRad,Hercules, Calif.). To this reaction mixture, 2 μl of diluted, frozen,and thawed samples were added and the plate sealed with a Microseal ‘B’Adhesive Seal (BioRad, Hercules, Calif.). The qPCR was performed on aBioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System(BioRad, Hercules, Calif.). The thermocycling conditions were 95° C. for15 minutes followed by 45 cycles of 95° C. for 5 seconds, 60° C. for 30seconds, and fluorescent readings of the FAM channel. Alternatively, theqPCR could be performed with other standard methods known to thoseskilled in the art.

The Cq value for each well on the FAM channel was determined by the CFXManager™ 3.0 software. The log 10(cfu/mL) of C. difficile eachexperimental sample was calculated by inputting a given sample's Cqvalue into a linear regression model generated from the standard curvecomparing the Cq values of the standard curve wells to the known log10(cfu/mL) of those samples. The log inhibition was calculated for eachsample by subtracting the log 10(cfu/mL) of C. difficile in the samplefrom the log 10(cfu/mL) of C. difficile in the sample on each assayplate used for the generation of the standard curve that has noadditional bacteria added. The mean log inhibition was calculated forall replicates for each composition.

A histogram of the range and standard deviation of each composition wasplotted. Ranges or standard deviations of the log inhibitions that weredistinct from the overall distribution were examined as possibleoutliers. If the removal of a single log inhibition datum from one ofthe binary pairs that were identified in the histograms would bring therange or standard deviation in line with those from the majority of thesamples, that datum was removed as an outlier, and the mean loginhibition was recalculated.

The pooled variance of all samples evaluated in the assay was estimatedas the average of the sample variances weighted by the sample's degreesof freedom. The pooled standard error was then calculated as the squareroot of the pooled variance divided by the square root of the number ofsamples. Confidence intervals for the null hypothesis were determined bymultiplying the pooled standard error to the z score corresponding to agiven percentage threshold. Mean log inhibitions outside the confidenceinterval were considered to be inhibitory if positive or stimulatory ifnegative with the percent confidence corresponding to the interval used.Samples with mean log inhibition greater than the 99% confidenceinterval (C.I) of the null hypothesis are reported as ++++, those with a95%<C.I.<99% as +++, those with a 90%<C.I.<95% as ++, those with a80%<C.I.<90% as + while samples with mean log inhibition less than thanthe 99% confidence interval (C.I) of the null hypothesis are reported as−−−−, those with a 95%<C.I.<99% as −−−, those with a 90%<C.I.<95% as −−,those with a 80%<C.I.<90% as −. Many binary pairs comprising KeystoneOTUs inhibit C. difficile as delineated in Table 20.

Assignment of a Network Classes Based on Phylogenetic Diversity andFunctional Modalities

“Network Classes” can be delineated by clustering computationallydetermined network ecologies into groupings based on the OTUs observedin a given network. In one example, OTUs are treated individualisticallywith each OTU representing a unique entity within the network. In otherexamples, the OTUs are clustered according to their phylogeneticrelationships defined by a phylogenetic tree, e.g., into clades. In yetanother embodiment, functional modules such as but not limited to KEGGOrthology Pathways can be used to cluster the networks, OTUs and Cladesaccording to the biological or biochemical functions they comprise. Aset of ecological networks from which a Network Class is defined, isselected using one or a combination of the following criteria: (i)networks that are derived from a biological phenotype, (ii) thefrequency at which a given network is observed across samples, or (iii)the size of the network. In one embodiment, the required thresholds forthe frequency at which a given network is observed across subjectsamples, and the size of a given network to be considered for furtheranalysis are defined by the 50^(th), 70^(th), 80^(th), or 90^(th)percentiles of the distribution of these variables. In anotherembodiment, the required thresholds are defined by the value for a givenvariable that is significantly different from the mean or median valuefor a given variable using standard parametric or non-parametricmeasures of statistical significance. In yet another embodiment,ecological networks derived from Network Classes are selected thatcontain 5 or fewer, 10 or fewer, 15 or fewer, 20 or fewer, 25 or fewer,or 50 or fewer OTUs, Clades, or Functional modalities.

Network Class ecologies are defined using a heatmap analytical strategywhereby the OTU content of a given network is mapped relative to thenetworks in which it exists (See, e.g., FIG. 18). FIG. 18 is aDerivation of Network Ecology Classes, according to an embodiment of theinvention. Subsets of networks are selected for use in defining NetworkClasses based on key biological criteria. Hierarchical Network clustersare defined by the presence (white) and absence (blue (or dark color))of OTUs and/or Functional Metabolic Pathways and Classes are defined asbranches of the hierarchical clustering tree based on the topologicaloverlap measure.

Both OTUs comprising the network ecologies and the network ecologiesthemselves are ordered using a dendrogram that represents therelatedness of each OTU to every other OTU, or each Network Ecology toevery other Network Ecology. The dendrogram for OTUs can be constructedusing various clustering algorithms including but not limited tophylogenetic maximum likelihood, hierarchical clustering, or k-meansclustering. In one embodiment, each row in the heatmap represents asingle OTU, each column represents a Network Ecology and the color inthe heatmap at a given row/column intersection represents whether thegiven OTU is present or absent in the given network. In anotherembodiment, the color in the heatmap represents the summed number ofoccurrences of the OTU in a set of related networks, represented as acluster in the dendrogram of network ecologies. In another embodiment,the row and column intersections represent a summary variable calculatedfrom the collapse of multiple rows and/or columns at selected nodes inthe dendrograms. Network Classes are defined finding significant branchpoints in the hierarchical dendrogram. In one embodiment these branchpoints are defined as branches of the hierarchical clustering tree basedon the topological overlap measure; this measure is a highly robustmeasure of network interconnectedness (Langfelder P, Zhang B, Horvath S.2008. Defining clusters from a hierarchical cluster tree: the DynamicTree Cut package for R. Bioinformatics 24: 719-720.). Network Classesare defined based on OTU presence/absence or presence/absence andfrequency patterns in network clusters; these patterns can be definedusing specific OTUs, taxonomic groupings, or phylogenetic clades definedby the phylogenetically derived dendrogram (i.e. phylogenetic tree).Network Classes can be defined with the intent of maximizing thephylogenetic diversity of the class, and/or representing specificregions of functional relevance on the phylogenetic tree.

We defined a set of Network Classes for the Network Ecologies reportedin Table 8 that were computationally inferred from health and diseasedatasets tied to CDAD studies using the method described above. Wedefined six Network Classes for these network ecologies (FIG. 18 andTables 12-13).

Example 7. Biologically-Informed Optimization of Network Ecologies Basedon Biological Properties

Network Ecologies can be optimized to have specific biologicalproperties including but not limited to being of specific size (asexample a specific number or OTUs); having a frequency of being observedin a population of healthy individuals (i.e. pervasiveness); having acertain percentage of spore forming OTUs as constituents; having acertain percentage of Keystone OTUs, clades or functions; having adefined phylogenetic breadth (as example defined by the totalevolutionary distance spanned on a tree by the constituent members, orby the total number of genera or other taxonomic unit); or comprisingspecific functional capabilities such as but not limited to the abilitymetabolize secondary bile acids, or produce short chain fatty acids(SCFAs), or the biological intersection in which network ecology fallsin a comparative phenotype map (see FIG. 19). The constituents of anetwork ecology can be optimized using both computational means as wellas experimental means.

In one embodiment, we developed a biopriority score for networks thatwas computationally derived. This algorithm took the form of[F1*W1]+[F2*W2]+[F3*W3]+[F4*W4] where F is a biological criteria ofinterest and W is a weighting for that factor based on its importance tothe derivation of the target network ecology. As example, if having anetwork with phylogenetic breadth was important one would weight thisfactor greater than the other factors. We developed a biopriority scorethat took into consideration the biological intersection of the network(FIG. 19), phylogenetic breadth, the pervasiveness or prevalence of thenetwork in populations of healthy individuals, and the percentage ofOTUs in the network that were Keystone OTUs. Network Ecologies reportedin Table 8 were ranked based on this scoring and networks with a highscore were preferentially screened and in vivo mouse model of C.difficile infection (Table 16).

In another embodiment we used a phylogenetic method paired withempirical testing to optimize the network ecologies for efficacy for thetreatment of CDAD. Based on computational insights from our networkanalysis (Table 8), applicants defined Keystone Clades that representspecific phylogenetic clusters of OTUs. Applicants constructed variousbacterial compositions using the methods described in Example 9 below,whereby applicants varied the phylogenetic breadth of the networkecologies based on the inclusion or exclusion of OTUs from specificclades. To test the effect of these variations on efficacy, 11 networksthat feature clade substitutions, additions, or subtractions were testedat the same target dose of 1e7 CFU per OTU per animal in the mouse modelof C. difficile infection experiment SP-376 (see Example 13 and Table16). FIG. 3 provides an overview of the various clade substitutions orremovals

The removal of clades 494 & 537 and the addition of clade_444 fromnetwork N1962, which was highly efficacious in protecting from symptomsof C. difficile infection with no mortality, yields network N1991, whichwas still largely protective of weight loss, but had increased meanmaximum clinical scores relative to N1962.

N1990 adds clades 444 & 478 to N1962, and resulted in decreased meanminimum relative weight and increased mean maximum clinical scoresrelative to N1962 while remaining efficacious relative to theexperiment's vehicle control.

Removal of clades 252 & 253 and the addition of clades 444 & 478 fromN1962 produces N1975, which has increased mortality, decreased meanminimum relative weight and increased mean maximum clinical scoresrelative to N1962, which is only slightly less efficacious than thevehicle control.

The optimization of network ecologies to design microbiome therapeutics(as example a composition comprised of bacterial OTUs) with particularbiological properties and features is executed using the strategy ofhaving a core Backbone Network Ecology onto which R-Groups are added orsubtracted to design toward particular characteristics. The Backboneforms a foundational composition of organisms or functions that are coreto efficacy and need be present to observe efficacy. On this backboneone can make various compositional modifications using R-groups.R-Groups can be defined in multiple terms including but not limited to:individual OTUs, individual or multiple OTUs derived from a specificphylogenetic clade, and functional modalities comprised of multiplefunctional pathways and/or specific metabolic pathways. In otherembodiments, R-groups could be considered prebiotics and otherco-factors that are design into, or administered with a network ecologyto promote specific biological properties.

Example 8. Network Analysis Across Multiple Data Sets and Selection ofTarget Network Ecologies with Capacity to Sporulate

One can select Network Ecologies and/or Network Class Ecologies as leadtargets by defining networks with a specific biological function oractivity such as sporulation. Networks Ecologies or Network ClassEcologies are first selected as described above and in Example 5 and 6.In one example, all Network Ecologies or Network Class Ecologies thatcontain at least one OTU that is capable of forming spores are targeted.In another example, all Network Ecologies or Network Class Ecologiesthat contain at least one OTU that is capable of forming spores, andthat are comprised of at least 50%, 75%, or 100% Keystone OTUs aretargeted. Keystone OTUs are selected as described above and in Example6. OTUs are defined as spore formers using either phenotypic assays (seee.g. Stackebrandt and Hippe. Taxonomy and Systematics. In Clostridia.Biotechnology and Medical Applications.) or genetic assays (see e.g.Abecasis A B, Serrano M, Alves R, Quintais L, Pereira-Leal J B, andHenriques A O. 2013. A genomic signature and the identification of newsporulation genes. J. Bacteriol.; Paredes-Sabja D, Setlow P, and SarkerM R. 2011. Germination of spores of Bacillales and Clostridialesspecies: mechanisms and proteins involved. Trends Microbiol. 19: 85-94).Exemplary network ecologies that are comprised of spore formers areillustrated in Tablell.

Example 9. Construction of Defined Ecobiotic Compositions

Source of Microbial Cultures. Pure cultures of organisms are isolatedfrom the stool, oral cavity or other niche of the body of clinicallyqualified donors (as in Example 10) that contains microorganisms ofinterest using microbiological methods including those described below,and as are known to those skilled in the art. Alternatively, purecultures are sourced from repositories such as the ATCC (atcc.org) orthe DSMZ (dsmz.de/) which preserve and distribute cultures of bacteria,yeasts, phages, cell lines and other biological materials.

Enrichment and Purification of Bacteria. To purify individual bacterialstrains, dilution plates were selected in which the density enablesdistinct separation of single colonies. Colonies were picked with asterile implement (either a sterile loop or toothpick) and re-streakedto BBA or other solid media. Plates were incubated at 37° C. for 3-7days. One or more well-isolated single colonies of the major morphologytype were re-streaked. This process was repeated at least three timesuntil a single, stable colony morphology is observed. The isolatedmicrobe was then cultured anaerobically in liquid media for 24 hours orlonger to obtain a pure culture of 106-1010 cfu/ml. Liquid growth mediummight include Brain Heart Infusion-based medium (Atlas, Handbook ofMicrobiological Media, 4th ed, ASM Press, 2010) supplemented with yeastextract, hemin, cysteine, and carbohydrates (for example, maltose,cellobiose, soluble starch) or other media described previously. Theculture was centrifuged at 10,000×g for 5 min to pellet the bacteria,the spent culture media was removed, and the bacteria were resuspendedin sterile PBS. Sterile 75% glycerol was added to a final concentrationof 20%. An aliquot of glycerol stock was titered by serial dilution andplating. The remainder of the stock was frozen on dry ice for 10-15 minand then placed at −80° C. for long term storage.

Cell Bank Preparation

Cell banks (RCBs) of bacterial strains were prepared as follows.Bacterial strains were struck from −80° C. frozen glycerol stocks toBrucella blood agar with Hemin or Vitamin K (Atlas, Handbook ofMicrobiological Media, 4th ed, ASM Press, 2010), M2GSC (Atlas, Handbookof Microbiological Media, 4th ed, ASM Press, 2010) or other solid growthmedia and incubated for 24 to 48 h at 37° C. in an anaerobic chamberwith a gas mixture of H2:CO2:N2 of 10:10:80. Single colonies were thenpicked and used to inoculate 250 ml to 1 L of Wilkins-Chalgren broth,Brain-Heart Infusion broth, M2GSC broth or other growth media, and grownto mid to late exponential phase or into the stationary phase of growth.Alternatively, the single colonies may be used to inoculate a pilotculture of 10 ml, which were then used to inoculate a large volumeculture. The growth media and the growth phase at harvest were selectedto enhance cell titer, sporulation (if desired) and phenotypes thatmight be associated desired in vitro or in vivo. Optionally, cultureswere grown static or shaking, depending which yielded maximal celltiter. The cultures were then concentrated 10 fold or more bycentrifugation at 5000 rpm for 20 min, and resuspended in sterilephosphate buffered saline (PBS) plus 15% glycerol. 1 ml aliquots weretransferred into 1.8 ml cryovials which were then frozen on dry ice andstored at −80° C. The identity of a given cell bank was confirmed by PCRamplification of the 16S rDNA gene, followed by Sanger direct cyclesequencing. See Examples 1, 2. Each bank was confirmed to yield coloniesof a single morphology upon streaking to Brucella blood agar or M2GSCagar. When more than one morphology was observed, colonies wereconfirmed to be the expected species by PCR and sequencing analysis ofthe 16S rDNA gene. Variant colony morphologies can be observed withinpure cultures, and in a variety of bacteria the mechanisms of varyingcolony morphologies have been well described (van der Woude, ClinicalMicrobiology Reviews, 17:518, 2004), including in Clostridium species(Wadsworth-KTL Anaerobic Bacteriology Manual, 6th Ed, Jousimie-Somer, etal 2002). For obligate anaerobes, RCBs were confirmed to lack aerobiccolony forming units at a limit of detection of 10 cfu/ml.

Titer Determination

The number of viable cells per ml was determined on the freshlyharvested, washed and concentrated culture by plating serial dilutionsof the RCB to Brucella blood agar or other solid media, and varied from106 to 1010 cfu/ml. The impact of freezing on viability was determinedby titering the banks after one or two freeze-thaw cycles on dry ice orat −80° C., followed by thawing in an anaerobic chamber at roomtemperature. Some strains displayed a 1-3 log drop in viable cfu/mlafter the 1st and/or 2nd freeze thaw, while the viability of others wereunaffected.

Preparation of Bacterial Compositions

Individual strains were typically thawed on ice and combined in ananaerobic chamber to create mixtures, followed by a second freeze at−80° C. to preserve the mixed samples. When making combinations ofstrains for in vitro or in vivo assays, the cfu in the final mixture wasestimated based on the second freeze-thaw titer of the individualstrains. For experiments in rodents, strains may be combined at equalcounts in order to deliver between 1e4 and 1e10 per strain.Additionally, some bacteria may not grow to sufficient titer to yieldcell banks that allowed the production of compositions where allbacteria were present at 1e10.

Selection of Media for Growth

Provided are appropriate media to support growth, including preferredcarbon sources. For example, some organisms prefer complex sugars suchas cellobiose over simple sugars. Examples of media used in theisolation of sporulating organisms include EYA, BHI, BHIS, and GAM (seebelow for complete names and references). Multiple dilutions are platedout to ensure that some plates will have well isolated colonies on themfor analysis, or alternatively plates with dense colonies may scrapedand suspended in PBS to generate a mixed diverse community.

Plates are incubated anaerobically or aerobically at 37° C. for 48-72 ormore hours, targeting anaerobic or aerobic spore formers, respectively.

Solid plate media include:

-   -   Gifu Anaerobic Medium (GAM, Nissui) without dextrose        supplemented with fructooligosaccharides/inulin (0.4%), mannitol        (0.4%), inulin (0.4%), or fructose (0.4%), or a combination        thereof    -   Sweet GAM [Gifu Anaerobic Medium (GAM, Nissui)] modified,        supplemented with glucose, cellobiose, maltose, L-arabinose,        fructose, fructooligosaccharides/inulin, mannitol and sodium        lactate)    -   Brucella Blood Agar (BBA, Atlas, Handbook of Microbiological        Media, 4th ed, ASM Press, 2010)    -   PEA sheep blood (Anaerobe Systems; 5% Sheep Blood Agar with        Phenylethyl Alcohol)    -   Egg Yolk Agar (EYA) (Atlas, Handbook of Microbiological Media,        4th ed, ASM Press, 2010)    -   Sulfite polymyxin milk agar (Mevissen-Verhage et al., J. Clin.        Microbiol. 25:285-289 (1987))    -   Mucin agar (Derrien et al., IJSEM 54: 1469-1476 (2004))    -   Polygalacturonate agar (Jensen & Canale-Parola, Appl. Environ.        Microbiol. 52:880-997 (1986))    -   M2GSC (Atlas, Handbook of Microbiological Media, 4th ed, ASM        Press, 2010)    -   M2 agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM        Press, 2010) supplemented with starch (1%), mannitol (0.4%),        lactate (1.5 g/L) or lactose (0.4%)    -   Sweet B—Brain Heart Infusion agar (Atlas, Handbook of        Microbiological Media, 4th ed, ASM Press, 2010) supplemented        with yeast extract (0.5%), hemin, cysteine (0.1%), maltose        (0.1%), cellobiose (0.1%), soluble starch (sigma, 1%), MOPS (50        mM, pH 7).    -   PY-salicin (peptone-yeast extract agar supplemented with        salicin) (Atlas, Handbook of Microbiological Media, 4th ed, ASM        Press, 2010).    -   Modified Brain Heart Infusion (M-BHI) [[sweet and sour]]        contains the following per L: 37.5 g Brain Heart Infusion powder        (Remel), 5 g yeast extract, 2.2 g meat extract, 1.2 g liver        extract, 1 g cystein HCl, 0.3 g sodium thioglycolate, 10 mg        hemin, 2 g soluble starch, 2 g FOS/Inulin, 1 g cellobiose, 1 g        L-arabinose, 1 g mannitol, 1 Na-lactate, 1 mL Tween 80, 0.6 g        MgSO4×7H2O, 0.6 g CaCl2, 6 g (NH4)2SO4, 3 g KH2PO4, 0.5 g        K2HPO4, 33 mM Acetic acid, 9 mM propionic acid, 1 mM Isobutyric        acid, 1 mM isovaleric acid, 15 g agar, and after autoclaving add        50 mL of 8% NaHCO₃ solution and 50 mL 1M MOPS-KOH (pH 7).    -   Noack-Blaut Eubacterium agar (See Noack et al. J. Nutr. (1998)        128:1385-1391)    -   BHIS az1/ge2—BHIS az/ge agar (Reeves et. al. Infect. Immun.        80:3786-3794 (2012)) [Brain Heart Infusion agar (Atlas, Handbook        of Microbiological Media, 4th ed, ASM Press, 2010) supplemented        with yeast extract 0.5%, cysteine 0.1%, 0.1% cellobiose, 0.1%        inulin, 0.1% maltose, aztreonam 1 mg/L, gentamycin 2 mg/L]    -   BHIS CInM az1/ge2—BHIS CInM [Brain Heart Infusion agar (Atlas,        Handbook of Microbiological Media, 4th ed, ASM Press, 2010)        supplemented with yeast extract 0.5%, cysteine 0.1%, 0.1%        cellobiose, 0.1% inulin, 0.1% maltose, aztreonam 1 mg/L,        gentamycin 2 mg/L].        Method of Preparing the Bacterial Composition for Administration        to a Patient

Two strains for the bacterial composition are independently cultured andmixed together before administration. Both strains are independently begrown at 37° C., pH 7, in a GMM or other animal-products-free medium,pre-reduced with 1 g/L cysteine ŸHCl. After each strain reaches asufficient biomass, it is preserved for banking by adding 15% glyceroland then frozen at −80° C. in 1 ml cryotubes.

Each strain is then be cultivated to a concentration of 1010 CFU/mL,then concentrated 20-fold by tangential flow microfiltration; the spentmedium is exchanged by diafiltering with a preservative mediumconsisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphatebuffer, or other suitable preservative medium. The suspension isfreeze-dried to a powder and titrated.

After drying, the powder is blended with microcrystalline cellulose andmagnesium stearate and formulated into a 250 mg gelatin capsulecontaining 10 mg of lyophilized powder (108 to 1011 bacteria), 160 mgmicrocrystalline cellulose, 77.5 mg gelatin, and 2.5 mg magnesiumstearate.

Example 10. Construction and Administration of an Ethanol-Treated SporePreparation

Provision of fecal material. Fresh fecal samples were obtained fromhealthy human donors who have been screened for general good health andfor the absence of infectious diseases, and meet inclusion and exclusioncriteria, inclusion criteria include being in good general health,without significant medical history, physical examination findings, orclinical laboratory abnormalities, regular bowel movements with stoolappearance typically Type 2, 3, 4, 5 or 6 on the Bristol Stool Scale,and having a BMI≥18 kg/m2 and ≤25 kg/m2. Exclusion criteria generallyincluded significant chronic or acute medical conditions includingrenal, hepatic, pulmonary, gastrointestinal, cardiovascular,genitourinary, endocrine, immunologic, metabolic, neurologic orhematological disease, a family history of, inflammatory bowel diseaseincluding Crohn's disease and ulcerative colitis, Irritable bowelsyndrome, colon, stomach or other gastrointestinal malignancies, orgastrointestinal polyposis syndromes, or recent use of yogurt orcommercial probiotic materials in which an organism(s) is a primarycomponent. Non-related donors were screened for general health historyfor absence of chronic medical conditions (including inflammatory boweldisease; irritable bowel syndrome; Celiac disease; or any history ofgastrointestinal malignancy or polyposis), absence of risk factors fortransmissible infections, antibiotic non-use in the previous 6 months,and negative results in laboratory assays for blood-borne pathogens(HIV, HTLV, HCV, HBV, CMV, HAV and Treponema pallidum) and fecalbacterial pathogens (Salmonella, Shigella, Yersinia, Campylobacter, E.coli 0157), ova and parasites, and other infectious agents (Giardia,Cryptosporidium, Cyclospora, Isospora) prior to stool donation. Sampleswere collected directly using a commode specimen collection system,which contains a plastic support placed on the toilet seat and acollection container that rests on the support. Feces were depositedinto the container, and the lid was then placed on the container andsealed tightly. The sample was then delivered on ice within 1-4 hoursfor processing. Samples were mixed with a sterile disposable tool, and2-4 g aliquots were weighed and placed into tubes and flash frozen in adry ice/ethanol bath. Aliquots are frozen at −80 degrees Celsius untiluse.

Optionally, the fecal material was suspended in a solution, and/orfibrous and/or particulate materials were removed using eitherfiltration or centrifugation. A frozen aliquot containing a known weightof feces was removed from storage at −80° C. and allowed to thaw at roomtemperature. Sterile 1×PBS was added to create a 10% w/v suspension, andvigorous vortexing was performed to suspend the fecal material until thematerial appeared homogeneous. The material was then left to sit for 10minutes at room temperature to sediment fibrous and particulate matter.The suspension above the sediment was then carefully removed into a newtube and contains a purified spore population. Optionally, thesuspension was then centrifuged at a low speed, e.g., 1000×g, for 5minutes to pellet particulate matter including fibers. The pellet wasdiscarded and the supernatant, which contained vegetative organisms andspores, was removed into a new tube. The supernatant was thencentrifuged at 6000×g for 10 minutes to pellet the vegetative organismsand spores. The pellet was then resuspended in 1×PBS with vigorousvortexing until the material appears homogenous.

Generation of a Spore Preparation from Alcohol Treatment of FecalMaterial

A 10% w/v suspension of human fecal material in PBS was filtered,centrifuged at low speed, and the supernate containing spores was mixedwith absolute ethanol in a 1:1 ratio and vortexed to mix. The suspensionwas incubated at room temperature for 1 hour. After incubation thesuspension was centrifuged at high speed to concentrate spores into apellet containing a purified spore-containing preparation. The supernatewas discarded and the pellet resuspended in an equal mass of glycerol,and the purified spore preparation was placed into capsules and storedat −80 degrees Celsius.

Characterization of Spores Content in Purified Spore Populations

In one embodiment, counts of viable spores are determined by performing10 fold serial dilutions in PBS and plating to Brucella Blood Agar Petriplates or applicable solid media. Plates are incubated at 37 degreesCelsius for 2 days. Colonies are counted from a dilution plate with50-400 colonies and used to back-calculate the number of viable sporesin the population. The ability to germinate into vegetative bacteria isalso demonstrated. Visual counts are determined by phase contrastmicroscopy. A spore preparation is either diluted in PBS or concentratedby centrifugation, and a 5 microliter aliquot is placed into a PetroffHauser counting chamber for visualization at 400× magnification. Sporesare counted within ten 0.05 mm×0.05 mm grids and an average spore countper grid is determined and used to calculate a spore count per ml ofpreparation. Lipopolysaccharide (LPS) reduction in purified sporepopulations is measured using a Limulus amebocyte lysate (LAL) assaysuch as the commercially available ToxinSensor™ Chromogenic LALEndotoxin Assay Kit (GenScript, Piscataway, N.J.) or other standardmethods known to those skilled in the art.

In a second embodiment, counts of spores are determined using a sporegermination assay. Germinating a spore fraction increases the number ofviable spores that will grow on various media types. To germinate apopulation of spores, the sample is moved to the anaerobic chamber,resuspended in prereduced PBS, mixed and incubated for 1 hour at 37° C.to allow for germination. Germinants can include amino-acids (e.g.,alanine, glycine), sugars (e.g., fructose), nucleosides (e.g., inosine),bile salts (e.g., cholate and taurocholate), metal cations (e.g., Mg2+,Ca2+), fatty acids, and long-chain alkyl amines (e.g., dodecylamine,Germination of bacterial spores with alkyl primary amines” J.Bacteriology, 1961.). Mixtures of these or more complex naturalmixtures, such as rumen fluid or Oxgall, can be used to inducegermination. Oxgall is dehydrated bovine bile composed of fatty acids,bile acids, inorganic salts, sulfates, bile pigments, cholesterol,mucin, lecithin, glycuronic acids, porphyrins, and urea. The germinationcan also be performed in a growth medium like prereduced BHIS/oxgallgermination medium, in which BHIS (Brain heart infusion powder (37 g/L),yeast extract (5 g/L), L-cysteine HCl (1 g/L)) provides peptides, aminoacids, inorganic ions and sugars in the complex BHI and yeast extractmixtures and Oxgall provides additional bile acid germinants.

In addition, pressure may be used to germinate spores. The selection ofgerminants can vary with the microbe being sought. Different speciesrequire different germinants and different isolates of the same speciescan require different germinants for optimal germination. Finally, it isimportant to dilute the mixture prior to plating because some germinantsare inhibitory to growth of the vegetative-state microorganisms. Forinstance, it has been shown that alkyl amines must be neutralized withanionic lipophiles in order to promote optimal growth. Bile acids canalso inhibit growth of some organisms despite promoting theirgermination, and must be diluted away prior to plating for viable cells.

For example, BHIS/oxgall solution is used as a germinant and contains0.5×BHIS medium with 0.25% oxgall (dehydrated bovine bile) where 1×BHISmedium contains the following per L of solution: 6 g Brain HeartInfusion from solids, 7 g peptic digest of animal tissue, 14.5 g ofpancreatic digest of casein, 5 g of yeast extract, 5 g sodium chloride,2 g glucose, 2.5 g disodium phosphate, and 1 g cysteine. Additionally,Ca-DPA is a germinant and contains 40 mM CaCl2, and 40 mM dipicolinicacid (DPA). Rumen fluid (Bar Diamond, Inc.) is also a germinant.Simulated gastric fluid (Ricca Chemical) is a germinant and is 0.2%(w/v) Sodium Chloride in 0.7% (v/v) Hydrochloric Acid. Mucin medium is agerminant and prepared by adding the following items to 1 L of distilledsterile water: 0.4 g KH2PO4, 0.53 g Na2HPO4, 0.3 g NH4Cl, 0.3 g NaCl,0.1 g MgCl2×6H2O, 0.11 g CaCl2, 1 ml alkaline trace element solution, 1ml acid trace element solution, 1 ml vitamin solution, 0.5 mg resazurin,4 g NaHCO3, 0.25 g Na2S×9 H2O. The trace element and vitamin solutionsprepared as described previously (Stams et al., 1993). All compoundswere autoclaved, except the vitamins, which were filter-sterilized. Thebasal medium was supplemented with 0.7% (v/v) clarified, sterile rumenfluid and 0.25% (v/v) commercial hog gastric mucin (Type III; Sigma),purified by ethanol precipitation as described previously (Miller &Hoskins, 1981). This medium is referred herein as mucin medium.

Fetal Bovine Serum (Gibco) can be used as a germinant and contains 5%FBS heat inactivated, in Phosphate Buffered Saline (PBS, FisherScientific) containing 0.137M Sodium Chloride, 0.0027M PotassiumChloride, 0.0119M Phosphate Buffer. Thioglycollate is a germinant asdescribed previously (Kamiya et al Journal of Medical Microbiology 1989)and contains 0.25M (pH10) sodium thioglycollate. Dodecylamine solutioncontaining 1 mM dodecylamine in PBS is a germinant. A sugar solution canbe used as a germinant and contains 0.2% fructose, 0.2% glucose, and0.2% mannitol. Amino acid solution can also be used as a germinant andcontains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mMproline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine. Agerminant mixture referred to herein as Germix 3 can be a germinant andcontains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mMproline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine, 0.2%taurocholate, 0.2% fructose, 0.2% mannitol, 0.2% glucose, 1 mM inosine,2.5 mM Ca-DPA, and 5 mM KCl. BHIS medium+DPA is a germinant mixture andcontains BHIS medium and 2 mM Ca-DPA. Escherichia coli spent mediumsupernatant referred to herein as EcSN is a germinant and is prepared bygrowing E. coli MG1655 in SweetB/Fos inulin medium anaerobically for 48hr, spinning down cells at 20,000 rcf for 20 minutes, collecting thesupernatant and heating to 60 C for 40 min. Finally, the solution isfilter sterilized and used as a germinant solution.

Determination of Bacterial Pathogens In Purified Spore Populations

Bacterial pathogens present in a purified spore population aredetermined by qPCR using specific oligonucleotide primers as follows.

Standard Curve Preparation

The standard curve is generated from wells containing the pathogen ofinterest at a known concentration or simultaneously quantified byselective spot plating. Serial dilutions of duplicate cultures areperformed in sterile phosphate-buffered saline. Genomic DNA is thenextracted from the standard curve samples along with the other samples.

Genomic DNA Extraction

Genomic DNA may be extracted from 100 μl of fecal samples, fecal-derivedsamples, or purified spore preparations using the Mo Bio Powersoil®-htp96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.)according to the manufacturer's instructions with two exceptions: thebeadbeating is performed for 2×4:40 minutes using a BioSpecMini-Beadbeater-96 (BioSpec Products, Bartlesville, Okla.) and the DNAis eluted in 50 μl of Solution C6. Alternatively the genomic DNA couldbe isolated using the Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), the Sigma-Aldrich Extract-N-Amp™ PlantPCR Kit, the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.)according to the manufacturer's instructions.

qPCR Composition and Conditions

The qPCR reaction to detect C. difficile contains 1× HotMasterMix(5PRIME, Gaithersburg, Md.), 900 nM of Wr-tcdB-F(AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG (SEQ ID NO. 2044), IDT, Coralville,Iowa), 900 nM of Wr-tcdB-R (CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO.2045), IDT, Coralville, Iowa), 250 nM of We-tcdB-P(6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO. 2046), LifeTechnologies, Grand Island, N.Y.), and PCR Water (Mo Bio Laboratories,Carlsbad, Calif.) to 18 μl (Primers adaped from: Wroblewski, D. et al.Rapid Molecular Characterization of Clostridium difficile and Assessmentof Populations of C. difficile in Stool Specimens. Journal of ClinicalMicrobiology 47:2142-2148 (2009)). This reaction mixture is aliquoted towells of a MicroAmp® Fast Optical 96-well Reaction Plate with Barcode(0.1 mL) (Life Technologies, Grand Island, N.Y.). To this reactionmixture, 2 μl of extracted genomic DNA is added. The qPCR is performedon a BioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-TimeSystem (BioRad, Hercules, Calif.). The thermocycling conditions are 95°C. for 2 minutes followed by 45 cycles of 95° C. for 3 seconds, 60° C.for 30 seconds, and fluorescent readings of the FAM and ROX channels.Other bacterial pathogens can be detected by using primers and a probespecific for the pathogen of interest.

Data Analysis

The Cq value for each well on the FAM channel is determined by the CFXManager™ Software Version 2.1. The log 10(cfu/ml) of each experimentalsample is calculated by inputting a given sample's Cq value into linearregression model generated from the standard curve comparing the Cqvalues of the standard curve wells to the known log 10(cfu/ml) of thosesamples. Viral pathogens present in a purified spore population aredetermined by qPCR as described herein and otherwise known in the art.

Example 11. Characterization of Microbiome in Ethanol-Treated SporePopulation and Patients Post-Treatment Microbial Population Engraftment,Augmentation, and Reduction of Pathogen Carriage in Patients Treatedwith Spore Compositions

Complementary genomic and microbiological methods were used tocharacterize the composition of the microbiota from Patient 1, 2, 3, 4,and 5, 6, 7, 8, 9, and 10 at pretreatment (pretreatment) and up to 4weeks post-treatment.

Table 3 shows bacterial OTUs associated with engraftment and ecologicalaugmentation and establishment of a more diverse microbial ecology inpatients treated with an ethanol-treated spore preparation. OTUs thatcomprise an augmented ecology are below the limit of detection in thepatient prior to treatment and/or exist at extremely low frequenciessuch that they do not comprise a significant fraction of the totalmicrobial carriage and are not detectable by genomic and/ormicrobiological assay methods in the bacterial composition. OTUs thatare members of the engrafting and augmented ecologies were identified bycharacterizing the OTUs that increase in their relative abundance posttreatment and that respectively are: (i) present in the ethanol-treatedspore preparation and not detectable in the patient pretreatment(engrafting OTUs), or (ii) absent in the ethanol-treated sporepreparation, but increase in their relative abundance in the patientthrough time post treatment with the preparation due to the formation offavorable growth conditions by the treatment (augmenting OTUs). Notably,the latter OTUs can grow from low frequency reservoirs in the patient,or be introduced from exogenous sources such as diet. OTUs that comprisea “core” augmented or engrafted ecology can be defined by the percentageof total patients in which they are observed to engraft and/or augment;the greater this percentage the more likely they are to be part of acore ecology responsible for catalyzing a shift away from a dysbioticecology. The dominant OTUs in an ecology can be identified using severalmethods including but not limited to defining the OTUs that have thegreatest relative abundance in either the augmented or engraftedecologies and defining a total relative abundance threshold. As example,the dominant OTUs in the augmented ecology of Patient-1 were identifiedby defining the OTUs with the greatest relative abundance, whichtogether comprise 60% of the microbial carriage in this patient'saugmented ecology by day 25 post-treatment.

Patient treatment with the ethanol-treated spore preparation leads tothe population of a microbial ecology that has greater diversity thanprior to treatment (See FIGS. 5 & 6). Genomic-based microbiomecharacterization confirmed engraftment of a range of OTUs that were notdetectable in the patient pretreatment (Table 3). These OTUs comprisedboth bacterial species that were capable and not capable of formingspores, and OTUs that represent multiple phylogenetic clades. Organismsnot detectable in Patient 1 pre-treatment either engraft directly fromthe ethanol-treated spore fraction or are augmented by the creation of agut environment favoring a healthy, diverse microbiota. Microbiologicalanalysis shows that Bacteroides fragilis group species were increased by4 and 6 logs in patients 1 and 2 (FIG. 7).

FIG. 5 shows the microbial diversity measured in the ethanol-treatedspore treatment sample and patient pre- and post-treatment samples.Total microbial diversity is defined using the Chaol Alpha-DiversityIndex and is measured at different genomic sampling depths to confirmadequate sequence coverage to assay the microbiome in the targetsamples. The patient pretreatment (purple) harbored a microbiome thatwas significantly reduced in total diversity as compared to theethanol-treated spore product (red) and patient post treatment at days 5(blue), 14 (orange), and 25 (green).

FIG. 6 shows patient microbial ecology is shifted by treatment with anethanol-treated spore treatment from a dysbiotic state to a state ofhealth. Principal Coordinates Analysis based on the total diversity andstructure of the microbiome (Bray-Curtis Beta-Diversity) of the patientpre- and post-treatment delineates that the engraftment of OTUs from thespore treatment and the augmentation of the patient microbial ecologyleads to a microbial ecology that is distinct from both the pretreatmentmicrobiome and the ecology of the ethanol-treated spore treatment (Table3).

FIG. 7 shows the augmentation of Bacteroides species in patients.Comparing the number of Bacteroides fragilis groups species in feces(cfu/g) pre-treatment and in week 4 post treatment reveals an increaseof 4 logs or greater. The ability of 16S-V4 OTU identification to assignan OTU as a specific species depends in part on the resolution of the16S-V4 region of the 16S gene for a particular species or group ofspecies. Both the density of available reference 16S sequences fordifferent regions of the tree as well as the inherent variability in the16S gene between different species will determine the definitiveness ofa taxonomic annotation to a given sequence read. Given the topologicalnature of a phylogenetic tree and that the tree represents hierarchicalrelationships of OTUs to one another based on their sequence similarityand an underlying evolutionary model, taxonomic annotations of a readcan be rolled up to a higher level using a clade-based assignmentprocedure (Table 1). Using this approach, clades are defined based onthe topology of a phylogenetic tree that is constructed from full-length16S sequences using maximum likelihood or other phylogenetic modelsfamiliar to individuals with ordinary skill in the art of phylogenetics.Clades are constructed to ensure that all OTUs in a given clade are: (i)within a specified number of bootstrap supported nodes from one another(generally, 1-5 bootstraps), and (ii) within a 5% genetic similarity.OTUs that are within the same clade can be distinguished as geneticallyand phylogenetically distinct from OTUs in a different clade based on16S-V4 sequence data. OTUs falling within the same clade areevolutionarily closely related and may or may not be distinguishablefrom one another using 16S-V4 sequence data. The power of clade basedanalysis is that members of the same clade, due to their evolutionaryrelatedness, play similar functional roles in a microbial ecology suchas that found in the human gut. Compositions substituting one specieswith another from the same clade are likely to have conserved ecologicalfunction and therefore are useful in the present invention.

Stool samples were aliquoted and resuspended 10× vol/wt in either 100%ethanol (for genomic characterization) or PBS containing 15% glycerol(for isolation of microbes) and then stored at −80° C. until needed foruse. For genomic 16S sequence analysis colonies picked from plateisolates had their full-length 16S sequence characterized as describedin Examples 2 and 3, and primary stool samples were prepared targetingthe 16S-V4 region using the method for heterogeneous samples describedherein.

Notably, 16S sequences of isolates of a given OTU are phylogeneticallyplaced within their respective clades despite that the actual taxonomicassignment of species and genus may suggest they are taxonomicallydistinct from other members of the clades in which they fall.Discrepancies between taxonomic names given to an OTU is based onmicrobiological characteristics versus genetic sequencing are known toexist from the literature. The OTUs footnoted in this table are known tobe discrepant between the different methods for assigning a taxonomicname.

Engraftment of OTUs from the ethanol-treated spore preparation treatmentinto the patient as well as the resulting augmentation of the residentmicrobiome led to a significant decrease in and elimination of thecarriage of pathogenic species other than C. difficile in the patient.16S-V4 sequencing of primary stool samples demonstrated that atpretreatment, 20% of reads were from the genus Klebsiella and anadditional 19% were assigned to the genus Fusobacterium. These strikingdata are evidence of a profoundly dysbiotic microbiota associated withrecurrent C. difficile infection and chronic antibiotic use. In healthyindividuals, Klebsiella is a resident of the human microbiome in onlyabout 2% of subjects based on an analysis of HMP database (hmpdacc.org),and the mean relative abundance of Klebsiella is only about 0.09% in thestool of these people. Its surprising presence at 20% relative abundancein Patient 1 before treatment is an indicator of a proinflammatory gutenvironment enabling a “pathobiont” to overgrow and outcompete thecommensal organisms normally found in the gut. Similarly, the dramaticovergrowth of Fusobacterium indicates a profoundly dysbiotic gutmicrobiota. One species of Fusobacterium, F. nucleatum (an OTUphylogenetically indistinguishable from Fusobacterium sp. 3_1_33 basedon 16S-V4), has been termed “an emerging gut pathogen” based on itsassociation with IBD, Crohn's disease, and colorectal cancer in humansand its demonstrated causative role in the development of colorectalcancer in animal models [Allen-Vercoe, Gut Microbes (2011) 2:294-8].Importantly, neither Klebsiella nor Fusobacterium was detected in the16S-V4 reads by Day 25 (Table 4).

To further characterize the colonization of the gut by Klebsiella andother Enterobacteriaceae and to speciate these organisms, pretreatmentand Day 25 fecal samples stored at −80 C as PBS-glycerol suspensionswere plated on a variety of selective media including MacConkey lactosemedia (selective for gram negative enterobacteria) and Simmons CitrateInositol media (selective for Klebsiella spp) [Van Cregten et al, J.Clin. Microbiol. (1984) 20: 936-41]. Enterobacteria identified in thepatient samples included K. pneumoniae, Klebsiella sp. Co_9935 and E.coli. Strikingly, each Klebsiella species was reduced by 2-4 logswhereas E. coli, a normal commensal organism present in a healthymicrobiota, was reduced by less than 1 log (Table 14 below). Thisdecrease in Klebsiella spp. carriage is consistent across multiplepatients. Four separate patients were evaluated for the presence ofKlebsiella spp. pre treatment and 4 weeks post treatment. Klebsiellaspp. were detected by growth on selective Simmons Citrate Inositol mediaas previously described. Serial dilution and plating, followed bydetermining cfu/mL titers of morphologically distinct species and 16Sfull length sequence identification of representatives of those distinctmorphological classes, allowed calculation of titers of specificspecies.

The genus Bacteroides is an important member of the gastrointestinalmicrobiota; 100% of stool samples from the Human Microbiome Projectcontain at least one species of Bacteroides with total relativeabundance in these samples ranging from 0.96% to 93.92% with a medianrelative abundance of 52.67% (hmpdacc.org reference data set HMSMCP).Bacteroides in the gut has been associated with amino acid fermentationand degradation of complex polysaccharides. Its presence in the gut isenhanced by diets rich in animal-derived products as found in thetypical western diet [David, L. A. et al, Nature (2013)doi:10.1038/nature12820]. Strikingly, prior to treatment, fewer than0.008% of the 16S-V4 reads from Patient 1 mapped to the genusBacteroides strongly suggesting that Bacteroides species were absent orthat viable Bacteroides were reduced to an extremely minor component ofthe patient's gut microbiome. Post treatment, ≥42% of the 16S-V4 readscould be assigned to the genus Bacteroides within 5 days of treatmentand by Day 25 post treatment 59.48% of the patients gut microbiome wascomprised of Bacteroides. These results were confirmed microbiologicallyby the absence of detectable Bacteroides in the pretreatment sampleplated on two different Bacteroides selective media: Bacteroides BileEsculin (BBE) agar which is selective for Bacteroides fragilis groupspecies [Livingston, S. J. et al J. Clin. Microbiol (1978). 7: 448-453]and Polyamine Free Arabinose (PFA) agar [Noack et al. J. Nutr. (1998)128: 1385-1391; modified by replacing glucose with arabinose]. Thehighly selective BBE agar had a limit of detection of <2×103 cfu/g,while the limit of detection for Bacteroides on PFA agar wasapproximately 2×107 cfu/g due to the growth of multiple non-Bacteroidesspecies in the pretreatment sample on that medium. Colony counts ofBacteroides species on Day 25 were up to 2×1010 cfu/g, consistent withthe 16S-V4 sequencing, demonstrating a profound reconstitution of thegut microbiota in Patient 1 (Table 5 below).

The significant abundance of Bacteroides in Patient 1 on Day 25 (and asearly as Day 5 as shown by 16S-V4 sequencing) is remarkable. ViableBacteroides fragilis group species were not present in theethanol-treated spore population based on microbiological plating (limitof detection of 10 cfu/ml). Thus, administration of the ethanol-treatedspore population to Patient 1 resulted in microbial population of thepatient's GI tract, not only due to the engraftment of bacterial speciessuch as but not limited to spore forming species, but also therestoration of high levels of non-spore forming species commonly foundin healthy individuals through the creation of a niche that allowed forthe repopulation of Bacteroides species. These organisms were mostlikely either present at extremely low abundance in the GI tract ofPatient 1, or present in a reservoir in the GI tract from which theycould rebound to high titer. Those species may also be reinoculated viaoral uptake from food following treatment. We term this healthyrepopulation of the gut with OTUs that are not present in the bacterialcomposition such as but not limited to a spore population orethanol-treated spore population, “Augmentation.” Augmentation is animportant phenomenon in that it shows the ability to use anethanol-treated spore ecology or other bacterial composition to restorea healthy microbiota by seeding a diverse array or commensal organismsbeyond the actual component organisms in the bacterial composition suchas but not limited to an ethanol-treated spore population itself;specifically the spore composition treatment itself and the engraftmentof OTUs from the spore composition create a niche that enables theoutgrowth of OTUs required to shift a dysbiotic microbiome to amicrobial ecology that is associated with health. The diversity ofBacteroides species and their approximate relative abundance in the gutof Patient 1 is shown in Table 16, comprising at least 8 differentspecies.

FIG. 8 shows species engrafting versus species augmenting in patientsmicrobiomes after treatment with a bacterial composition such as but notlimited to an ethanol-treated spore population. Relative abundance ofspecies that engrafted or augmented as described were determined basedon the number of 16S sequence reads. Each plot is from a differentpatient treated with the bacterial composition such as but not limitedto an ethanol-treated spore population for recurrent C. difficile.

The impact of the bacterial composition such as but not limited to anethanol-treated spore population treatment on carriage of imipenemresistant Enterobacteriaceae was assessed by plating pretreatment andDay 28 clinical samples from Patients 2, 4 and 5 on MacConkey lactoseplus 1 ug/mL of imipenem. Resistant organisms were scored by morphology,enumerated and DNA was submitted for full length 16S rDNA sequencing asdescribed above. Isolates were identified as Morganella morganii,Providencia rettgeri and Proteus penneri. Each of these are gutcommensal organisms; overgrowth can lead to bacteremia and/or urinarytract infections requiring aggressive antibiotic treatment and, in somecases, hospitalization [Kim, B-N, et al Scan J. Inf Dis (2003) 35:98-103; Lee, I—K and Liu, J-W J. Microbiol Immunol Infect (2006) 39:328-334; O'Hara et al, Clin Microbiol Rev (2000) 13: 534]. The titer oforganisms at pretreatment and Day 28 by patient is shown in Table 17.Importantly, administration of the bacterial composition such as but notlimited to an ethanol-treated spore preparation resulted in greater than100-fold reduction in 4 of 5 cases of Enterobacteriaceae carriage withmultiple imipenem resistant organisms (See Table 17 which shows titers(in cfu/g) of imipenem-resistant M. morganii, P. rettgeri and P. pennerifrom Patients 2, 4 & 5).

In addition to speciation and enumeration, multiple isolates of eachorganism from Patient 4 were grown overnight in 96-well trays containinga 2-fold dilution series of imipenem in order to quantitativelydetermine the minimum inhibitory concentration (MIC) of antibiotic.Growth of organisms was detected by light scattering at 600 nm on aSpectraMax M5e plate reader. In the clinical setting, these species areconsidered resistant to imipenem if they have an MIC of 1 ug/mL orgreater. M. morganii isolates from pretreatment samples from Patient 4had MICs of 2-4 ug/mL and P. penneri isolates had MICs of 4-8 ug/mL.Thus, the bacterial composition, such as but not limited to, anethanol-treated spores administered to Patient 4 caused the clearance of2 imipenem resistant organisms (Table 4). While this examplespecifically uses a spore preparation, the methods herein describe howone skilled in the art would use a more general bacterial composition toachieve the same effects. The specific example should not be viewed as alimitation of the scope of this disclosure.

Identifying the Core Ecology From the Bacterial Combination

Ten different bacterial compositions were made by the ethanol-treatedspore preparation methods from 6 different donors (as described above).The spore preparations were used to treat 10 patients, each sufferingfrom recurrent C. difficile infection. Donors were identified using theinclusion/exclusion criteria described above under provision of fecalmaterial. None of the patients experienced a relapse of C. difficile inthe 4 weeks of follow up after treatment, whereas the literature wouldpredict that 70-80% of subjects would experience a relapse followingcessation of antibiotic [Van Nood, et al, NEJM (2013)]. Thus, theethanol-treated spore preparations derived from multiple differentdonors and donations showed remarkable clinical efficacy. Theseethanol-treated spore preparations are a subset of the bacterialcompositions described herein and the results should not be viewed as alimitation on the scope of the broader set of bacterial compositions.

To define the Core Ecology underlying the remarkable clinical efficacyof the bacterial compositions e.g. ethanol-treated spore preparations,the following analysis was carried out. The OTU composition of the sporepreparation was determined by 16S-V4 rDNA sequencing and computationalassignment of OTUs per Example 2. A requirement to detect at least tensequence reads in the ethanol-treated spore preparation was set as aconservative threshold to define only OTUs that were highly unlikely toarise from errors during amplification or sequencing. Methods routinelyemployed by those familiar to the art of genomic-based microbiomecharacterization use a read relative abundance threshold of 0.005% (seee.g. Bokulich, A. et al. 2013. Quality-filtering vastly improvesdiversity estimates from Illumina amplicon sequencing. Nature Methods10: 57-59), which would equate to ≥2 reads given the sequencing depthobtained for the samples analyzed in this example, as cut-off which issubstantially lower than the ≥10 reads used in this analysis. Alltaxonomic and clade assignments were made for each OTU as described inExample 2. The resulting list of OTUs, clade assignments, and frequencyof detection in the spore preparations are shown in Table 18. Table 18shows OTUs detected by a minimum of ten 16S-V4 sequence reads in atleast one ethanol-treated spore preparatio. OTUs that engraft in atreated patients and the percentage of patients in which they engraftare denoted, as are the clades, spore forming status, and Keystone OTUstatus. Starred OTUs occur in ≥80% of the ethanol preps and engraft in≥50% of the treated patients.

Next, it was reasoned that for an OTU to be considered a member of theCore Ecology of the bacterial composition, that OTU must be shown toengraft in a patient. Engraftment is important for two reasons. First,engraftment is a sine qua non of the mechanism to reshape the microbiomeand eliminate C. difficile colonization. OTUs that engraft with higherfrequency are highly likely to be a component of the Core Ecology of thespore preparation or broadly speaking a set bacterial composition.Second, OTUs detected by sequencing a bacterial composition (as in Table6 may include non-viable cells or other contaminant DNA molecules notassociated with the composition. The requirement that an OTU must beshown to engraft in the patient eliminates OTUs that representnon-viable cells or contaminating sequences. Table 6 also identifies allOTUs detected in the bacterial composition that also were shown toengraft in at least one patient post-treatment. OTUs that are present ina large percentage of the bacterial composition e.g. ethanol sporepreparations analyzed and that engraft in a large number of patientsrepresent a subset of the Core Ecology that are highly likely tocatalyze the shift from a dysbiotic disease ecology to a healthymicrobiome.

A third lens was applied to further refine insights into the CoreEcology of the bacterial composition (e.g. spore preparation).Computational-based, network analysis has enabled the description ofmicrobial ecologies that are present in the microbiota of a broadpopulation of healthy individuals (see Example 5). These networkecologies are comprised of multiple OTUs, some of which are defined asKeystone OTUs. Keystone OTUs are computationally defined as described inExample 6. Keystone OTUs form a foundation to the microbially ecologiesin that they are found and as such are central to the function ofnetwork ecologies in healthy subjects. Keystone OTUs associated withmicrobial ecologies associated with healthy subjects are often aremissing or exist at reduced levels in subjects with disease. KeystoneOTUs may exist in low, moderate, or high abundance in subjects. Table 6further notes which of the OTUs in the bacterial composition e.g. sporepreparation are Keystone OTUs exclusively associated with individualsthat are healthy and do not harbor disease. The presence ofcomputationally derived Keystone OTUs in the Core Ecology of the dosesvalidates the predictive capacity of computationally derived networkecologies.

There are several important findings from this data. A relatively smallnumber of species, 16 in total, are detected in all of the sporepreparations from 6 donors and 10 donations. This is surprising becausethe HMP database (hmpdacc.org) describes the enormous variability ofcommensal species across healthy individuals. The presence of a smallnumber of consistent OTUs lends support to the concept of a Core Ecologyand Backbone Networks. The engraftment data further supports thisconclusion. A regression analysis shows a significant correlationbetween frequency of detection in a spore preparation and frequency ofengraftment in a donor: R=0.43 (p<0.001). While this may seem obvious,there is no a priori requirement that an OTU detected frequently in thebacterial composition e.g. spore preparation will or should engraft. Forinstance, Lutispora thermophila, a spore former found in all ten sporepreparations, did not engraft in any of the patients. Bilophilawadsworthia, a gram negative anaerobe, is present in 9 of 10 donations,yet it does not engraft in any patient, indicating that it is likely anon-viable contaminant in the ethanol-treated spore preparation.Finally, it is worth noting the high preponderance of previously definedKeystone OTUs among the most frequent OTUs in the spore preparations.

These three factors—prevalence in the bacterial composition such as butnot limited to a spore preparation, frequency of engraftment, anddesignation as a Keystone OTUs—enabled the creation of a “Core EcologyScore” (CES) to rank individual OTUs. CES was defined as follows:

40% weighting for presence of OTU in spore preparation

-   -   multiplier of 1 for presence in 1-3 spore preparations    -   multiplier of 2.5 for presence in 4-8 spore preparations    -   multiplier of 5 for presences in ≥9 spore preparations

40% weighting for engraftment in a patient

-   -   multiplier of 1 for engraftment in 1-4 patients    -   multiplier of 2.5 for engraftment in 5-6 patients    -   multiplier of 5 for engraftment in ≥7 patients

20% weighting to Keystone OTUs

-   -   multiplier of 1 for a Keystone OTU    -   multiplier of 0 for a non-Keystone OTU

Using this guide, the CES has a maximum possible score of 5 and aminimum possible score of 0.8. As an example, an OTU found in 8 of the10 bacterial composition such as but not limited to a spore preparationsthat engrafted in 3 patients and was a Keystone OTU would be assignedthe follow CES:CES=(0.4×2.5)+(0.4×1)+(0.2×1)=1.6

Table 7 ranks the top 20 OTUs by CES with the further requirement thatan OTU must be shown to engraft to be a considered an element of a coreecology.

Defining Efficacious Subsets of the Core Ecology

The number of organisms in the human gastrointestinal tract, as well asthe diversity between healthy individuals, is indicative of thefunctional redundancy of a healthy gut microbiome ecology (see The HumanMicrobiome Consortia. 2012. Structure, function and diversity of thehealthy human microbiome. Nature 486: 207-214). This redundancy makes ithighly likely that subsets of the Core Ecology describe therapeuticallybeneficial components of the bacterial composition such as but notlimited to an ethanol-treated spore preparation and that such subsetsmay themselves be useful compositions for populating the GI tract andfor the treatment of C. difficile infection given the ecologiesfunctional characteristics. Using the CES, individual OTUs can beprioritized for evaluation as an efficacious subset of the Core Ecology.

Another aspect of functional redundancy is that evolutionarily relatedorganisms (i.e. those close to one another on the phylogenetic tree,e.g. those grouped into a single clade) will also be effectivesubstitutes in the Core Ecology or a subset thereof for treating C.difficile.

To one skilled in the art, the selection of appropriate OTU subsets fortesting in vitro (see Example 20 below) or in vivo (see Examples 13 or14) is straightforward. Subsets may be selected by picking any 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 OTUs from Table 6, with a particularemphasis on those with higher CES, such as the OTUs described Table 7.In addition, using the clade relationships defined in Example 2 aboveand Table 1, related OTUs can be selected as substitutes for OTUs withacceptable CES values. These organisms can be cultured anaerobically invitro using the appropriate media (selected from those described inExample 5 above), and then combined in a desired ratio. A typicalexperiment in the mouse C. difficile model utilizes at least 10⁴ andpreferably at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or more than 10⁹ colonyforming units of a each microbe in the composition. Variations in theculture yields may sometimes mean that organisms are combined in unequalratios, e.g. 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000, or greater than1:100,000. What is important in these compositions is that each strainbe provided in a minimum amount so that the strain's contribution to theefficacy of the Core Ecology subset can be measured. Using theprinciples and instructions described here, it is straightforward forone of skill in the art to make clade-based substitutions to test theefficacy of subsets of the Core Ecology. Table 18 describes the cladesfor each OTU detected in a spore preparation, and Table 1 describes theOTUs that can be used for substitutions based on clade relationships.Examples of network ecologies empirically screened in vivo are presentedin Example 13 below.

Example 12. Presence of Network Ecologies and Keystone OTUs inClinically Prepped Ethanol-Treated Spore Preparation and CDAD PatientsPost Treatment

Network ecologies computationally determined as described in Example 5and reported in Table 8 as being networks or subsets of networkscharacteristic of health states in the context of CDAD or other diseaseindications (Table 14a-b) are observed in the ethanol-treated sporepreparation (a.k.a. the bacterial composition) and the microbiome ofpatients post treatment (see Example 11) indicating that they play animportant role in treatment of CDAD and other indications. For eachcomputationally determined network ecology (Table 8), we determinedwhether the full network or a subset of the network was observed in themicrobiome of (i) each of the 10 ethanol-treated spore preparations usedto treat patients with recurrent Clostridium difficile associateddiarrhea; (ii) the engrafted ecology of each of the 10 patients (seeExample 11); (iii) the augmented ecology of each of the 10 patients (seeExample 11); or (iv) of each of the 10 patient's microbiomepretreatment. If the computationally determined networks are indeedrepresentative of a state of health and not a disease state, one wouldexpect that these networks would be responsible for catalyzing a shiftfrom a disease state to a health state. This can happen either by thenetwork ecology changing the gut environment to favor the growth of OTUsthat are required to establish a health state (i.e. promotingaugmentation) or by the engraftment of OTUs in the bacterial compositionor both. Applicants observed that numerous computationally determinednetworks and/or subsets of these networks were in fact observed both inthe bacterial composition used to treat the patients and the microbiotathat expanded post-treatment (Table 14b). These same networks orsub-sets of networks were significantly under-represented in thepatients pre-treatment. To demonstrate this, we computed the percentageof network OTUs that are found in (i) the treatment bacterialcomposition, (ii) the post-treatment augmented ecology, (iii) thepost-treatment engrafted ecology, and (iv) the pretreatment ecology(i.e. patient microbiome prior to administration of the bacterialcomposition). Applicants observed across all doses of bacterialcomposition and patient samples that on average 46%±19%, 28%±14%,11%±8%, and 7%±4% of the computed networks OTUs were present in thevarious microbiome ecologies, respectively (reported here asaverage±standard deviation). There was a significant difference(p<0.0001, ANOVA) between all of these percentages indicating that priorto treatment, the OTUs found in CDAD patients are significantlyunder-represented in the networks, and that the network OTUs aresignificantly over-represented in the bacterial compositions andpost-treatment patient samples, affirming the predictive utility of thecomputational network analysis. These results in combination with thosereported in Table 14b demonstrate that, prior to treatment, the patientsharbored a significantly lower number of OTUs that comprised networkecologies. In contrast, the ecology of the bacterial composition, aswell as the augmenting ecologies whose appearance was catalyzed by thespore population, were significantly overrepresented in patients whoseCDAD resolved due to treatment.

We observed both large and small computationally determined networkecologies characteristic of states of health in the ethanol-treatedspore population and the patients post treatment (Table 14a). Theseobserved networks ranged in size from 2-15 OTUs and were comprised ofOTUs that represented from 29% to 100% of the OTUs in thecomputationally determined network ecology. Notably, on average thenetwork ecologies found in the ethanol-treated spore population or thepatient ecologies post treatment comprised 72%±15% (average±SD) of thecomputationally determined network ecology again strongly indicating animportant role of the computed network ecologies in catalyzing a shiftin a dysbiotic disease ecology to a state of health in these patientswith recurrent CDAD. Further, Keystone OTUs in the computationallydetermined network ecologies were frequently observed in theethanol-treated spore preparations and in the patients' post-treatmentgut ecologies. Clades representing Keystone OTUs where typically morecommon in the bacterial composition and post-treatment patient ecologiesthan in the pre-treatment dysbiotic patient ecology (Table 15).

The computed network ecologies and their respective subsets that areobserved in the ethanol-treated spore preparation and the variouspatient ecologies post-treatment represent both complete andfoundational networks (e.g., Backbone Network Ecology). Microbialtherapeutics can be comprised of these network ecologies in theirentirety, or they can be modified by the addition or subtraction ofother OTUs or functional modalities as described in Example 7 andExample 22 to design particular phylogenetic and/or functionalcharacteristics, including metabolic functions such as SCFA productionor bile acid metabolism, into the microbial therapeutic.

Example 13. In Vivo Validation of Network Ecology Bacterial CompositionsEfficacy in Clostridium Difficile Infection Prevention Mouse Model

To test the therapeutic potential of the bacterial composition such asbut not limited to a spore population, a prophylactic mouse model of C.difficile infection was used (model based on Chen X, Katchar K,Goldsmith J D, Nanthakumar N, Cheknis A, Gerding D N, Kelly C P. 2008. Amouse model of Clostridium difficile-associated disease.Gastroenterology 135: 1984-1992.). Two cages of five mice each weretested for each arm of the experiment. All mice received an antibioticcocktail consisting of 10% glucose, kanamycin (0.5 mg/ml), gentamicin(0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269 mg/ml),ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) and Vancomycin(0.056 mg/ml) in their drinking water on days −14 through −5 and a doseof 10 mg/kg Clindamycin by oral gavage on day −3. On day −1, testarticles are spun for 5 minutes at 12,100 rcf, their supernatants'removed, and the remaining pellets are resuspended in sterile PBS,prereduced if bacterial composition is not in spore form, and deliveredvia oral gavage. On day 0 they were challenged by administration ofapproximately 4.5 log 10 cfu of C. difficile (ATCC 43255) or sterile PBS(for the Naive arm) via oral gavage. Optionally a positive control groupreceived vancomycin from day −1 through day 3 in addition to theantibiotic protocol and C. difficile challenge specified above. Feceswere collected from the cages for analysis of bacterial carriage.Mortality, weight and clinical scoring of C. difficile symptoms basedupon a 0-4 scale by combining scores for Appearance (0-2 pts based onnormal, hunched, piloerection, or lethargic), and Clinical Signs (0-2points based on normal, wet tail, cold-to-the-touch, or isolation fromother animals) are assessed every day from day −2 through day 6. Meanminimum weight relative to day −1 and mean maximum clinical score wherea death is assigned a clinical score of 4 as well as average cumulativemortality are calculated. Reduced mortality, increased mean minimumweight relative to day −1, and reduced mean maximum clinical score withdeath assigned to a score of 4 relative to the vehicle control are usedto assess the success of the test article.

Table 16 reports results for 15 experiments of the prophylactic mousemodel of C. difficile infection. In the 15 experiments, 157 of the armstested network ecologies, with 86 distinct networks ecologies tested(Table 17). Of those 157 arms, 136 of the arms and 73 of the networksperformed better than the respective experiment's vehicle control arm byat least one of the following metrics: cumulative mortality, meanminimum relative weight, and mean maximum clinical score. Examples ofefficacious networks include but are not limited to networks N1979 astested in SP-361 which had 0% cumulative mortality, 0.97 mean minimumrelative weight, and 0 mean maximum clinical score or N2007 which had10% cumulative mortality, 0.91 mean minimum relative weight, and 0.9mean maximum clinical score with both networks compared to the vehiclecontrol in SP-361 which had 30% cumulative mortality, 0.88 mean minimumrelative weight, and 2.4 mean maximum clinical score. In SP-376, N1962had no cumulative mortality, mean maximum clinical scores of 0 at bothtarget doses tested with mean minimum relative weights of 0.98 and 0.95for target doses of 1e8 and 1e7 CFU/OTU/mouse respectively. Theseresults confirm that bacterial compositions comprising bacteriaidentified from computationally determined networks or subsets of thesedetermined networks have utility and efficacy in the mouse model.

Example 14. In Vivo Validation of Network Ecology Bacterial CompositionEfficacy in Prophylactic and Relapse Prevention Hamster Model

Previous studies with hamsters using toxigenic and nontoxigenic strainsof C. difficile demonstrated the utility of the hamster model inexamining relapse post antibiotic treatment and the effects ofprophylaxis treatments with cecal flora in C. difficile infection(Wilson et al. 1981, Wilson et al. 1983, Borriello et al. 1985) and morebroadly in gastrointestinal infectious disease. To demonstrateprophylactic use of ethanol-treated spores and ethanol treated,gradient-purified spores to ameliorate C. difficile infection, thefollowing hamster model was used. In the prophylactic model, Clindamycin(10 mg/kg s.c.) was given on day −5, the test article or control wasadministered on day −3, and C. difficile challenge occurred on day 0. Inthe positive control arm, vancomycin was then administered on day 1-5(and vehicle control was delivered on day −3). Feces were collected onday −5, −4, −1, 1, 3, 5, 7, 9 and fecal samples were assessed forpathogen carriage and reduction by microbiological methods. 16Ssequencing approaches or other methods could also be utilized by oneskilled in the art. Mortality was assessed multiple times per daythrough 21 days post C. difficile challenge. The percentage survivalcurves showed that ethanol-treated spores and ethanol treated,gradient-purified spores better protected the hamsters compared to theVancomycin control, and vehicle control.

FIG. 9 shows a prophylaxis model with the ethanol-treated sporepreparation and the ethanol treated, gradient-purified sporepreparation. In the relapse prevention model, hamsters were challengedwith toxigenic C. difficile strains on day 0, and treated withclindamycin by oral gavage on day 1, and vancomycin was dosed on days2-6. Test or control treatment was then administered on day 7, 8, and 9.The groups of hamsters for each arm consisted of 8 hamsters per group.Fecal material was collected on day −1, 1, 3, 5, 7, 10 and 13 andhamster mortality was assessed throughout. Survival curves were used toassess the efficacy of the test articles, e.g., ethanol treated orethanol treated, gradient purified spores versus the control treatmentin preventing hamster death. The survival curves demonstrated maximumefficacy for the ethanol treated, gradient-purified spores followed bythe ethanol-treated spores. Both treatments improved survival percentageover vancomycin treatment.

Also in the relapse prevention model, the efficacy of a bacterialcommunity of pure cultures, N1962, was tested. The survival curvesdemonstrate protection against relapse by N1962 relative to thevancomycin control treatment.

FIG. 10 shows a relapse prevention model with ethanol-treated spores andethanol treated, gradient purified spores. In particular, it shows an invivo hamster Clostridium difficile relapse prevention model to validateefficacy of ethanol-treated spores and ethanol treated, gradientpurified spores.

FIG. 11 shows a relapse prevention model with a bacterial community. Inparticular, it shows an in vivo hamster Clostridium difficile relapseprevention model to validate efficacy of network ecology bacterialcomposition.

Example 15. Derivation of Functional Profile of Individual MicrobialOTUs or Consortia of OTUs Representing Specific Network Ecologies

To generate a functional profile of an OTU, or consortium of OTUs onecan leverage multiple-omic data types. These include, but are notlimited to functional prediction based on 16S rRNA sequence, functionalannotation of metagenomic or full-genome sequences, transcriptomics, andmetabolomics. A consortium of OTUs of interest can be defined usingnumerous criteria including but not limited to: (i) a computationallyderived network of OTUs based on the analysis of samples that representstates of health and disease such as those delineated in Example 5 andreported in Table 8, (ii) a consortia of OTUs that are identified in anindividual sample or group of samples using either a 16S-based,metagenomic-based, or microbiological-based methods such as delineatedin Examples 3, 4 and 16, and (iii) a list of OTUs derived from theassessment of literature.

For 16S rRNA sequences, phylogenetic investigation of communities byreconstruction of unobserved states, also known as PICRUSt (Langille M GI, Zaneveld J, Caporaso J G, McDonald D, Knights D, Reyes J A, ClementeJ C, Burkepile D E, Vega Thurber R L, Knight R, et al. 2013. Predictivefunctional profiling of microbial communities using 16S rRNA marker genesequences. Nat Biotechnol.), enables the prediction of a functionalmetabolic pathway of an OTU or a consortium of OTUs based on the KEGGdatabase of reference functional pathways and functional ontologies(Kyoto Encyclopedia of Genes and Genomes; genome.jp/kegg/). PICRUStmatches the taxonomic annotation of a single 16S sequence read with areference functional annotation of a genome sequence for a given OTU orset of OTUs. From these reference genome annotations, a functionalannotation is assigned to each OTU. PICRUSt is composed of twohigh-level workflows: gene content inference and metagenome inference.The gene content inference produces gene content predictions for a setof reference OTUs as well as copy number predictions. The metagenomeinference then uses these inputs and an OTU table that defines the OTUsin a sample and their relative abundances to then infer the functionalmetabolic profile of the OTUs in the OTU table. In an alternative, butrelated method, one can lookup for all of the OTUs in a consortia theOTU taxonomic identifications in a functional reference database such asIMG (http://img.jgi.doe.gov) and then derive a functional annotation ofthe network by concatenating the database's metabolic pathway maps (e.g.KEGG Pathway Orthology in case of IMG) for each of the OTUs in theconsortia (see below for specific example).

To generate functional annotation from metagenomic or whole genomeshotgun sequence data, reads are first clustered and then representativereads are annotated. Sequence annotation is then performed as describedin Example 1, with the additional step that sequences are eitherclustered or assembled prior to annotation. Following sequencecharacterization as described above using a technology such as but notlimited to Illumina, sequence reads are demultiplexed using the indexingbarcodes. Following demultiplexing sequence reads are clustered using arapid clustering algorithm such as but not limited to UCLUST(drive5.com/usearch/manual/uclust algo.html) or hash-based methods suchVICUNA (Xiao Yang, Patrick Charlebois, Sante Gnerre, Matthew G Coole,Niall J. Lennon, Joshua Z. Levin, James Qu, Elizabeth M. Ryan, MichaelC. Zody, and Matthew R. Henn. 2012. De novo assembly of highly diverseviral populations. BMC Genomics 13:475). Following clustering arepresentative read for each cluster is identified and analyzed asdescribed above in Example 2 “Primary Read Annotation”. The result ofthe primary annotation is then applied to all reads in a given cluster.In another embodiment, metagenomic sequences are first assembled intocontigs and then these assembled contigs are annotated using methodsfamiliar to one with ordinary skill in the art of genome assembly andannotation. Platforms such as but not limited to MetAMOS (TJ. Treangenet al. 2013 Geneome Biology 14:R2), and HUMAaN (Abubucker S, Segata N,Goll J, Schubert A M, Izard J, Cantarel B L, Rodriguez-Mueller B, ZuckerJ, Thiagaraj an M, Henrissat B, et al. 2012. Metabolic Reconstructionfor Metagenomic Data and Its Application to the Human Microbiome ed. J.A. Eisen. PLoS Computational Biology 8: e1002358) are suitable foranalysis of metagenomic data sets using the methods described above.Tools such as MetAMOS are also suitable for the generation of afunctional annotation of complete genome sequence assembled from thesample or obtained from a reference genome database such as but notlimited to NCBI's genome database (ncbi.nlm.nih.gov/genome). In allcases, functional pathways are derived from the sequence readannotations based on the mapping of the sequence annotations to afunctional database, such as but not limited to KEGG (genome.jp/kegg),Biocyc (biocyc.org), IMG (img.jgi.doe.gov), MetaCyc (metacyc.org), orReactome (reactome.org). Various tools are available for this task thatare familiar to one with ordinary skill in the art including, but notlimited to, The HMP Unified Metabolic Analysis Network (HUMAnN)(Abubucker S, Segata N, Goll J, Schubert A M, Izard J, Cantarel B L,Rodriguez-Mueller B, Zucker J, Thiagarajan M, Henrissat B, et al. 2012.Metabolic Reconstruction for Metagenomic Data and Its Application to theHuman Microbiome ed. J.A. Eisen. PLoS Computational Biology 8:e1002358). The HUMAnN software recovers the presence, absence, andabundance of microbial gene families and pathways from metagenomic data.Cleaned short DNA reads are aligned to the KEGG Orthology (or any othercharacterized sequence database with functional annotation assigned togenetic sequences) using accelerated translated BLAST. Gene familyabundances are calculated as weighted sums of the alignments from eachread, normalized by gene length and alignment quality. Pathwayreconstruction is performed using a maximum parsimony approach followedby taxonomic limitation (to remove false positive pathwayidentifications) and gap filling (to account for rare genes in abundantpathways). The resulting output is a set of matrices of pathwaycoverages (presence/absence) and abundances, as analyzed here for theseven primary body sites of the Human Microbiome Project.

Transcriptomic or RNA-Seq data are also a means to generate a functionalprofile of a sample (Wang Z, Gerstein M, Snyder M. 2009. RNA-Seq: arevolutionary tool for transcriptomics. Nat Rev Genet 10: 57-63).Briefly, long RNAs are first converted into a library of cDNA fragmentsthrough either RNA fragmentation or DNA fragmentation. Sequencingadaptors appropriate to the sequencing technology being used fordownstream sequencing are subsequently added to each cDNA fragment and ashort sequence is obtained from each cDNA using high-throughputsequencing technology. The resulting sequence reads are aligned with thereference genome or transcriptome and annotated and mapped to functionalpathways as described above. Reads are categorized as three types:exonic reads, junction reads and poly(A) end-reads. These three types ofreads in combination with the gene annotation are used to generate abase-resolution expression profile for each gene.

In yet another method to generate a metabolic profile of a microbialecology, characterization of metabolites produced by the ecology areanalyzed in tissues or fluids. Samples can include, without limitation,blood, urine, serum, feces, ileal fluid, gastric fluid, pulmonaryaspirates, tissue culture fluid, or bacterial culture supernatants. Bothtargeted and untargeted methods can be utilized for metabolomicsanalysis (Patti G J, Yanes O, Siuzdak G. 2012. Innovation: Metabolomics:the apogee of the omics trilogy. Nat Rev Mol Cell Biol 13: 263-269.).Metabolomic methods utilize LC/MS-based technologies to generate ametabolite profile of sample. In the triple quadrupole (QqQ)-basedtargeted metabolomic workflow, standard compounds for the metabolites ofinterest are first used to set up selected reaction monitoring methods.Here, optimal instrument voltages are determined and response curves aregenerated using reference standards for absolute quantification. Afterthe targeted methods have been established on the basis of standardmetabolites, metabolites are extracted from the sample using methodsfamiliar to one with ordinary skill in the art. Extraction methods caninclude liquid:liquid extraction using organic solvents or two-phaseaqueous methods, solid phase extraction using hydrophobic or ionexchange resins, filtrations to remove solid contaminants,centrifugation or other means of clarification, and counter-currenttechniques. The data output provides quantification only of thosemetabolites for which standards are available. In the untargetedmetabolomic workflow, extracted metabolites are first iseparated byliquid chromatography followed by mass spectrometry (LC/MS). After dataacquisition, the results are processed by using bioinformatic softwaresuch as XCMS to perform nonlinear retention time alignment and identifypeaks that are changing between the groups of related samples. The m/zvalues for the peaks of interest are searched in a metabolite databasesto obtain putative identifications. Putative identifications are thenconfirmed by comparing tandem mass spectrometry (MS/MS) data andretention time data to that of standard compounds. The untargetedworkflow is global in scope and outputs data related to comprehensivecellular metabolism.

Applicants generated a functional profile for all of the computationallydetermined network ecologies delineated in Table 8 and Table 14a thatwere derived using the methods outlined in Example 5. Table 18 and Table21 provide written description of the corresponding functional networkecologies respectively. For each network, applicants generated afunctional metabolic profile by concatenating the KEGG OrthologyPathways for each OTU available in the IMG functional database(img.jgi.doe.gov). The taxonomic annotations of each OTU in the networkwere mapped to the taxon_display_names in the IMG database. For eachtaxon_display_name the taxon_iod with the best 16S sequence match to the16S sequence of the OTU in the computed network ecology was selected(best match based on expectation value and an alignment score). Thefunctional annotation for each OTU in the network was then derived fromIMG's KEGG Orthology Pathway (i.e. ko_id) for the given taxon_iod. KEGGOrthology Pathways (KO) for all the OTUs in the network wereconcatenated and then the list was made unique to generate anon-redundant functional profile of the network. In another embodiment,the ko_id list is not made unique and the functional profile of thenetwork is defined based on the relative abundances of the ko_ids notjust their presence or absence. It is with the level of ordinary skillusing the aforementioned disclosure to construct functional networkecologies that substitute the exemplified OTUs with equivalent OTUs thatharbor the orthologous KEGG Orthology Pathways. Such substitiutions arecontemplated to be within the scope of the present invention, eitherliterally or as an equivalent to the claimed invention as determined bydetermined by a court of competent jurisdiction.

Each functional network ecology was scored for its ability to metabolizebile acids and to produce short chain fatty acids (SCFAs). As describedabove, both bile acid metabolism and the production of SCFAs bybacterial ecologies plays an important role in human health.Specifically, applicants subsetted the KEGG Orthology Pathways computedfor each network ecology to those described to be involved in secondarybile acid biosynthesis, butryrate (a.k.a. butanoate) metabolism,propionate (a.k.a. propanoate) metabolism, or pyruvate metabolism (leadsto production of acetate). We identified and ranked network ecologiesfor their capacity to metabolize bile acid and produce SCFAs by defininga bile acid and SCFA functional score (F-Score) that defines a networkecologies' capacity to perform these two important metabolic roles. TheF-score is defined by the total number of KEGG Orthology Pathways in agiven network that mapped to secondary bile acid biosynthesis, abutyrate metabolism, a propionate metabolism, or a pyruvate metabolism(Table 18). A functional translation of the KEGG Orthology Pathways(i.e., KO numbers) and their respective metabolic ontologyclassification is provided in Table 19 as reference. Significantly, asshown in Table 18, there are only two computed network ecologies thatdid not harbor at least one pathway related to secondary bile acidbiosynthesis, butyrate metabolism, propionate metabolism, or a pyruvatemetabolism, suggesting both likely importance of these pathways to themetabolism of a large number of gastrointestinal ecologies, and theimportance of these pathways to catalyzing a shift from a disease to ahealth state in the example cases of CDAD and Type 2 Diabetes.

Example 16. Use of Biolog Assay to Generate a Nutrient UtilizationFunctional Profile of an OTU or Consortium of OTU

Metabolic capabilities of individual organisms or a consortia oforganisms can be determined using Biolog technology in which metabolicactivity is detected by measurement of NADH production using a redoxsensitive dye. Carbon source or other metabolic capabilities of a singlespecies can be determined, as described below. Carbon source utilizationof an ecology or network can also be assessed using the same methods.

A screen was performed to test the ability of Clostridium difficile andpotential competitor species to utilize a panel of 190 different carbonsources. The screen was carried out using PM1 and PM2 MicroPlates(Biolog #12111, #12112), IF-0a base media (Biolog #72268) and BiologRedox Dye Mix D (Biolog #74224). For each strain, a 1 uL aliquot from−80° C. glycerol stock was streaked out for single colonies to solidBrucella Blood Agar plates (BBA) (Anaerobe Systems #AS-111) andincubated anaerobically at 37° C. for 24 hr. A single colony was thenre-streaked to a BBA plate and incubated anaerobically at 37° C. for 24hr. The MicroPlates were pre-reduced by incubating for at least 24 hr ina hydrogen free anaerobic environment before use. All liquid media andsupplements used were pre-reduced by placing them in an anaerobicchamber with loose lids for at least 24 hr before use. Alternatively,combinations of bacteria can also be tested.

The base media for inoculation was prepared by adding 0.029 mL of 1Mpotassium ferricyanide to 0.240 mL of Dye Mix D followed by addition of19.7 mL of IF-0a, 4 mL sterile water and 0.024 mL 0.5 mM menadione. Forsome species, the concentrations of potassium ferricyanide and menadionewere adjusted to achieve the optimal redox balance or to test multipleredox conditions. Potassium ferricyanide was tested at a finalconcentration of 0.38, 0.12, 0.038 and 0.06 mM. Menadione was tested ata final concentration of 0.5, 0.16 and 0.05 μM. In total, this yields 9redox conditions for testing. Reduction of the tetrazolium dye thatforms the basis for the endpoint measurement was sensitive to the redoxstate of each bacterial culture, and thus to the ratio of menadione topotassium ferricyanide. It was therefore important to test variousratios for each bacterial isolate and was also important in some casesto test a species at multiple menadione/potassium ferricyanide ratios inorder to detect all conditions in which a possible nutrient utilizationwas detectable. Some species were tested beyond the 20 hr time point todetect all conditions resulting in a positive result. In these casesplates were read at 20, 44 or 96 hr.

Using a sterile, 1 μL microbiological loop, a loopful of biomass wasscraped from the BBA plate and resuspended in the base media byvortexing. The OD was adjusted to 0.1 at 600 nm using a SpectraMax M5plate reader. The bacterial suspension was then aliquoted into each wellof the PM1 and PM2 plates (100 μL per well). The plates were incubatedat 37° C. for 20 hr in a rectangular anaerobic jar (Mitsubishi) with 3anaerobic, hydrogen-free gas packs (Mitsubishi AnaeroPack). After 20 hr,OD at 550 nm was read using a SpectraMax M5 plate reader. Wells werescored as a weak hit if the value was 1.5× above the negative controlwell, and a strong hit if the value was 2× above the negative controlwell. The results are shown in the Table in FIG. 4.

The following list of nutrient sources were tested: L-Arabinose,N-Acetyl-D-Glucosamine, D-Saccharic Acid, SuccinicAcid, D-Galactose,L-AsparticAcid, L-Proline, D-Alanine, D-Trehalose, D-Mannose, Dulcitol,D-Serine, D-Sorbitol, Glycerol, L-Fucose, D-Glucuronic Acid, D-GluconicAcid, D, L-alpha-Glycerol-Phosphate, D-Xylose, L-Lactic Acid, FormicAcid, D-Mannitol, L-Glutamic Acid, D-Glucose-6-Phosphate, D-GalactonicAcid-gamma-Lactone, D,L-Malic Acid, D-Ribose, Tween 20, L-Rhamnose,D-Fructose, Acetic Acid, alpha-D-Glucose, Maltose, D-Mellibiose,Thymidine, L-Asparagine, D-Aspartic Acid, D-Glucosaminic Acid,1,2-Propanediol, Tween 40, alpha-Keto-Glutaric Acid,alpha-Keto-ButyricAcid, alpha-Methyl-D-Galactoside, alpha-D-Lactose,Lactulose, Sucrose, Uridine, L-Glutamine, M-Tartaric Acid,D-Glucose-1-Phosphate, D-Fructose-6-Phosphate, Tween 80,alpha-Hydroxy-Glutaric-gamma-lactone, alpha-Hydroxy Butyric Acid,beta-Methyl-D-Glucoside, Adonitol, Maltotriose, 2-Deoxy Adenosine,Adenosine, Glycyl-L-Aspartic Acid, Citric Acid, M-Inositol, D-Threonine,Fumaric Acid, Bromo Succinic Acid, Propionic Acid, Mucic Acid, GlycolicAcid, Glyoxylic Acid, D-Cellobiose, Inosine, Glycyl-L-Glutamic Acid,Tricarballylic Acid,L-Serine, L-Threonine, L-Alanine, L-Alanyl-Glycine,Acetoacetic Acid, N-Acetyl-beta-D-Mannosamine, Mono Methyl Succinate,Methyl Pyruvate, D-Malic Acid, L-Malic Acid,Glycyl-L-Proline, p-HydroxyPhenyl Acetic Acid, m-Hydroxy Phenyl Acetic Acid, Tyramine, D-Psicose,L-Lyxose, Glucuronamide, Pyruvic Acid, L-GalactonicAcid-gamma-Lactone,D-Galacturonic Acid,Pheylethyl-amine,2-aminoethanol,Chondroitin Sulfate C, alpha-Cyclodextrin, beta-Cyclodextrin,gamma-Cyclodextrin, Dextrin, Gelatin, Glycogen, Inulin, Laminarin,Mannan, Pectin, N-Acetyl-D-Galactosamine, N-Acetyl-Neuramic Acid,beta-D-Allose, Amygdalin, D-Arabinose, D-Arabitol, L-Arabitol, Arbutin,2-Deoxy-D-Ribose, I-Erythritol, D-Fucose,3-0-beta-D-Galacto-pyranosyl-D-Arabinose, Gentibiose, L-Glucose,Lactitol, D-Melezitose, Maltitol, alpha-Methyl-D-Glucoside,beta-Methyl-D-Galactoside, 3-Methyl Glucose,beta-Methyl-D-GlucoronicAcid, alpha-Methyl-D-Mannoside,beta-Metyl-D-Xyloside, Palatinose, D-Raffinose, Salicin, Sedoheptulosan,L-Sorbose, Stachyose, D-Tagatose, Turanose, Xylitol,N-Acetyl-D-Glucosaminitol, gamma-Amino Butyric Acid, delta-Amino ValericAcid, Butyric Acid, Capric Acid, Caproic Acid, Citraconic Acid,Citramalic Acid, D-Glucosamine, 2-Hydroxy Benzoic Acid, 4-HydroxyBenzoic Acid, beta-Hydroxy Butyric Acid, gamma-Hydroxy Butyric Acid,alpha-Keto Valeric Acid, Itaconic Acid, 5-Keto-D-Gluconic Acid, D-LacticAcid Methyl Ester, Malonic Acid, Melibionic Acid, Oxalic Acid,Oxalomalic Acid, Quinic Acid, D-Ribino-1,4-Lacton, Sebacic Acid, SorbicAcid, Succinamic Acid, D-Tartaric Acid, L-Tartaric Acid, Acetamide,L-Alaninamide, N-Acetyl-L-Glutamic Acid, L-Arginine, Glycine,L-Histidine, L-Homserine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine,L-Lysine, L-Methionine, L-Ornithine, L-Phenylalanine, L-PyroglutamicAcid, L-Valine, D,L-Carnithine, Sec-Butylamine, D,L-Octopamine,Putrescine, Dihydroxy Acetone, 2,3-Butanediol, 2,3-Butanone, 3-Hydroxy2-Butanone.

Additionally, one of skill in the art could design nutrient utilizationassays for a broader set of nutrients using the methods described aboveincluding complex polysaccharides or prebiotics.

A similar screen can be performed to test the utilization of vitamins,amino acids, or cofactors. In these instances, Biolog MicroPlates forscreening of vitamins, amino acids or cofactors that are of interestwould be used in place of the PM1 and PM2 plates, for example PMS. Table2 contains a list of representative vitamins, minerals, and cofactors.For each strain tested, a universal carbon source such as glucose willbe used as a positive control to demonstrate reduction of thetetrazolium dye under the specific conditions of the assay.

Example 17. In Vitro Screening of Microbes for 7-AlphadehydroxylaseActivity

Cultures of individual microbes are grown overnight and frozen for lateruse as described according to Example 9. The sodium salts of CA, CDCA,GCA, GCDCA, TCA, and TCDCA (Sigma) are obtained and prepared as aqueousstock solutions. For initial screening to define organisms capable of7-alphadehydroxylation reactions, growth media are prepared containing0.4 mM of each bile salt. Cultures are inoculated from a 1:100 dilutionof the frozen stock into the media and grown in an anaerobic chamber for24-48 hours, or until the culture is turbid. Two mL of culture isacidified by the addition of 1 mL of 2N HCl and 100 ug of23-nordeoxycholic acid (Steraloids) as an internal reference standard.The acidified mixture is extracted twice with 6 mL of diethyl ether. Theorganic extracts are combined and then evaporated and derivatized tomethyl esters with diazomethane. Gas chromatography is performed on a 7ft (ca. 2 m) 3% OV-1 column at 260° C. and a 3% OV-17 column at 250° C.after trimethylsilylation of the methylated bile acids with Tri-Sil(Pierce, Rockford, Ill.). The retention times of the silylated bileacids are compared with those of reference products representing CA,CDCA, DCA and LCA.

For strains showing 7-alphadehydroxylase activity, a kinetic assessmentis performed by harvesting a growing culture of each organisms ofinterest, washing and resuspending in fresh media at a concentration ofbetween 108 to 1010 cfu/mL. The sodium salts of CA, CDCA, GCA, GCDCA,TCA, and TCDCA are then added at 0.5 to 5 mM and the resulting cultureis sampled at 1, 2, 4 and 8 hours. The sample is analyzed as describedabove to find organisms with maximal activity. Highly active strains areselected for further incorporation into microbial compositions thatexhibit maximal 7-alphadehydroxylase activity.

Example 18. In Vitro Screening of Microbes for Bile Salt HydrolaseActivity

Cultures of individual microbes are grown overnight and frozen for lateruse as described according to Example 9. The sodium salts of GCA, GCDCA,TCA, and TCDCA (Sigma) are obtained and prepared as aqueous stocksolutions. Overnight, actively growing cultures are combined with 0.5 to5 mM of conjugated bile acid and allowed to incubate for 24-48 hours. Toanalyze cultures, 0.5 mL of culture is first centrifuged at 3,000×g for10 min to remove the bacteria, and is then acidified with 5 uL of 6 NHCl. This acidified supernatant is combined with an equal volume ofmethanol containing 4 mM of 23-nordeoxycholic as an internal standard.The samples are vortexed for at least 2 min and clarified bycentrifugation at 1000×g for 15 min. Samples are filtered through a 0.2um filter prior to HPLC analysis according to the method described byJones et al2003 J Med Sci 23: 277-80. Briefly, the isocratic method isperformed on a reversed-phase C-18 column (LiChrosorb RP-18, 5 m,250×4.6 mm from HiChrom, Novato, Calif., USA). Acetate buffer isprepared daily with 0.5 M sodium acetate, adjusted to pH 4.3 witho-phosphoric acid, and filtered through a 0.22 m filter. The flow is 1.0mL/min and the detection is performed at 205 nm. The injection loop isset to 20 uL.

For strains showing bile salt hydrolase activity, a kinetic assessmentis performed by harvesting a growing culture of each organisms ofinterest, washing and resuspending in fresh media at a concentration ofbetween 108 to 1010 cfu/mL. The sodium salts of GCA, GCDCA, TCA, andTCDCA are then added at 0.5 to 5 mM and the resulting culture is sampledat 1, 2, 4 and 8 hours. The sample is analyzed by HPLC as describedabove to find organisms with maximal activity. Highly active strains areselected for further incorporation into microbial compositions thatexhibit maximal bile salt hydrolase activity.

Example 19. In vitro Screening of Microbial Communities for7-alpha-dehydroxylase Activity

Measurement of the conversion of 7-alpha-hydroxyl bile salts (primarybile salts) to 7-dehydroxy-bile salts (secondary bile salts) by singlebacterial strains or bacterial communities is determined in an in vitroassay, and can be used to screen a library of organisms, wholecommunities or subsets of communities using limiting dilutions toidentify simpler compositions. Communities to be studies include cecalor fecal communities from animals with altered gastrointestinalmicrobiota due to antibiotics, diet, genetics, enterohepatic metabolism,or other experimental perturbations that cause GI alterations, or fromhuman fecal samples from healthy individuals or those with alteredgastrointestinal microbiomes due to antibiotics, diet, enterohepaticmetabolism dysfunction, metabolic dysfunction, or gastrointestinalinfection. Dilutions or subsets of these communities (such as could begenerated by selective culturing for of the whole community to enrichfor aerobes, anaerobes, Gram positives, Gram negatives, spore formers orusing other microbiological selections known to one skilled in the art)can be utilized to identify a group of organisms required for aparticular multi-step conversion.

To assay 7-alpha-dehydroxylation activity in vitro, an enzymatic assayis established to quantify the amount of 7-alphahydroxy bile acid in asample. Recombinant 7-alpha-hydroxysteroid dehydrogenase (7-alpha-HSDH)from E. coli (MyBiosource.com) is an enzyme that oxidizes the 7-hydroxygroup to a ketone and simultaneously reduces NAD+ to NADH+H+. Theproduction of NADH is monitored at 340 nm using the extinctionco-efficient of 6.2×103 M-1 cm-1.

A community of microbes is prepared according to Example 9 or,alternatively a preparation of cecal or fecal bacteria from mice or fromhuman feces or a dilution thereof, or an enriched community thereof, canbe tested after being washed 5 times to remove bile acids from thematrix. To the initial sample, a mixture of one or more primary bileacids including but not limited to CA, CDCA or any of their taurine orglycine conjugates is added to a final total concentration of 0.5-5 mM.An initial 100 uL aliquot is removed and heated at 55° C. for 15 min toquench further enzymatic activity. The bacterial composition is thenincubated under anaerobic conditions at 37° C., and aliquots are removedsequentially after 30 min, 1 hour, 2 hours, 4 and 8 hours and heated asper above. An assay mix is prepared by combining 0.9 mL glycine-NaOHbuffer pH 9.5, 50 uL of 53 mM NAD+ (Sigma), and 20 uL of freshlyprepared 7-alpha-HSDH (4 mg/mL in distilled water). 80 uL of assay mixis combined with 20 uL of each aliquot in a 96-well microtiter plate andincubated at 37° C. on a SpectraMax m5 plate reader, monitoring A340.The incubation is allowed to proceed until the A340 value achieves itsmaximum. Total 7-alpha-hydroxyl bile acid is determined using theextinction coefficient for NADH. Changes due to dehydroxylation by thebacterial composition are calculated by subtracting the final value atany timepoint from the initial value.

Microbial communities of interest can be further fractionated usingmethods described in Example 9.

Example 20. In Vitro Evaluation of Mixed Microbial Cultures for BileAcid Metabolism

Candidates strains identified in Examples 17, 18 and 19 above are testedusing the methods defined for bile salt hydrolase activity and7-alphadehydroxylase activity are combined in communities to evaluatesynergies among strains and define ecologies for further testing inanimal models. Synergies include: i) the potential for more rapidconversion from conjugated primary bile salts to unconjugated,dehydryoxylated bile acids; ii) the potential for a broader range ofproducts than determined by the additive combination of activities; iii)equivalent activity at a lower concentration (cfu) of the individualstrains. Combinations exhibiting such synergies are particularly favoredfor subsequent in vivo testing. Another important function of acommunity is to remove endproducts of a microbial conversion so as toavoid inhibition of growth through product accumulation. For bile acidconversion, communities can optionally include organisms capable ofdegrading taurine, using it both as a carbon and nitrogen source andusing the sulfonic acid group as an electron acceptor in fermentation.

Example 21. Combinations of Bacterial Compositions For SCFA ProductionUnder Variable Conditions

Combinations of synergistic bacterial compositions may be selected suchthat the composition is capable of producing SCFA under a wide range ofin vitro conditions when the entire mixture is tested together. That is,a combination of bacterial compositions comprises multiple pairs oforganisms that, together with a complex carbon source, are capable ofsynthesizing SCFA. Combinations may be constructed that are capable ofproducing a given set of SCFAs, for example butyrate and proprionate,but not acetate, or that produce butyrate, proprionate and acetate, butthat the acetate is then used by another organism as a carbon source. Anumber of specific combinations of final SCFAs may be generated bycommunities designed by one skilled in the art. Construction ofbacterial combinations follows the protocol described in Example 9.

Example 22. De Novo Design of Network Ecologies with Specific FunctionalProperties

The role of the microbiome in mediating and influencing human metabolicfunction is well established. Microbes produce secondary bile acids (asexample, Louis P, Flint H J. 2009. Diversity, metabolism and microbialecology of butyrate-producing bacteria from the human large intestine.FEMS Microbiol Lett 294: 1-8.), short chain fatty acids (for example,Smith P M et al. 2013 Science. The microbial metabolites, short-chainfatty acid regulate colonic Treg cell homeostasis 341: 569-73) as wellas numerous other functional metabolites that influence immunity andmetabolic health of the human host.

To identify consortia of microbes suitable for the use as therapeutics,to influence host metabolic functions, and to treat microbial dysbiosisone can computationally derive in silico network ecologies that possessspecific metabolic functions such as, but not limited to, a single ormultiple metabolic nodes in the functional pathways involved insecondary bile acid biosynthesis (FIG. 12), butyrate metabolism (FIG.13), propionate metabolism (FIG. 14), or pyruvate metabolism (FIG. 15).As additional examples, network ecologies can be in silico designed totarget host genes involved in important host:microbe innate and adaptiveimmune responses through targets such as the Toll-like receptors (TLRs)and nucleotide-binding oligomerization domains (NOD) (Saleh M,Trinchieri G. 2011. Innate immune mechanisms of colitis andcolitis-associated colorectal cancer. Nat Rev Immunol 11: 9-20. andKnight P, Campbell B J, Rhodes J M. 2008. Host-bacteria interaction ininflammatory bowel disease. Br Med Bull 88: 95-113.). In addition, thefunctional pathways to target for in silico network ecology design canbe empirically defined by comparing the microbiomes of samples derivedfrom different phenotypes such as but not limited to a state of diseaseand a state of health. For example, one can compare the microbiome andcorresponding metabolic functional profile of individuals with andwithout insulin resistance. Vrieze et al. have shown that treatment withvancomycin can reduce the diversity of the microbiome and result in asmall, but statistically significant change in peripheral insulinsensitivity. Similar changes are not observed following amoxicillintreatment (Vrieze, A et al., 2013, J Hepatol. Impact of oral vancomycinon gut microbiota, bile acid metabolism, and insulin sensitivitydx.doi.org/10.1016/j.jhep.2013.11.034). Decreased insulin sensitivitywas associated with a decrease in the presence of secondary bile acidsDCA, LCA and iso-LCA and an increase in primary bile acids CA and CDCAincreased. In another example, Applicants can compare the microbiome andmetabolomic profile of healthy individuals to those with CDAD disease.In yet another example, Applicants can compare the microbiome andmetabolomic profile of healthy individuals to those that harbor IBD,IBS, Ulcerative Colitis, Crohn's Disease, Type-2-Diabetes, orType-1-Diabetes.

For both CDAD and insulin resistance, Applicants can define thefunctional metabolic profile of the respective disease and healthmicrobiomes using the 16S and metagenomic genomic methods outlined inExample 15. In another embodiment, Applicants can use the transcriptomicand metabolomic methods outlined in Example 15. In another embodiment weuse functional metabolic information garnered from the literature andderived from functional screens such as but not limited to BiologMicroPlates (see Example 16). From these profiles, Applicants cangenerate a metabolic function matrix for both the disease state and thehealth state. This matrix is comprised of columns of OTUs and rows ofKEGG Orthology Pathways delineated as described in Example 15. Ametabolic function matrix can be generated for both the disease stateand the health state. From these disease and health matrices, Applicantscan compute a delta-function matrix (i.e. difference matrix) thatdefines the OTUs, the relative abundance of the metabolic pathways theyharbor, and the difference in the relative abundance between the diseaseand health state. In another embodiment, the relative abundances arediscretized to be a binary, ternary, or quaternary factor. Thisdelta-function matrix defines the differences in the microbiomedistinguishing the disease state from the health state.

One can then design a network ecology with the desired functionalcharacteristics described by the delta-function matrix. In oneembodiment, one can use a greedy algorithm to optimize for the mostparsimonious solution to the delta-function matrix. One can designtowards (i.e. select) the minimal number of OTUs to capture the fullbreadth of KEGG Orthology Pathways that are represented in the healthstate. In short, the greedy algorithm repetitively samples the OTUsspanning the greatest number of health associated KEGGs until thedesired breath of KEGGs is obtained to define a functional networkecology comprised of specific OTUs. In another embodiment, one canoptimize the greedy algorithm to weigh OTUs that are from specificphylogenetic clades. In another embodiment, one can start with thecomputationally derived network ecologies derived using the methodsdefined in Example 5 to both seed and constrain the greedy algorithm toreturn functional network ecologies that embody the co-occurrencerelationships that exist between OTUs. Microbial therapeuticcompositions comprised of the OTUs of the computed network ecologies areconstructed using the methods defined in Example 9. In one embodiment,constraints around network ecologies are defined by networks found inspecific individuals. In another embodiment, strains of each OTU thatare used for construction preferentially are selected from strainsisolated from the same individual since these strains are evolutionaryco-evolved and have an increased likelihood of functional synergy.

In another embodiment, Applicants computationally defined in silico anetwork ecology with the explicit capacity to produce butyrate. In thisembodiment, Applicants defined the health state in terms of themetabolic pathways and associated gene products required for themetabolism of non-digestable carbohydrates via fermentation by colonicbacteria and by the gene products leading from mono- and di-saccharidesand simple substrates such as acetate and lactate to butyrate (FIGS.12-15). We then used the IMG functional database(http://img.jgi.doe.gov) of OTU KEGG Orthology Pathways (i.e. ko_id) togenerate a metabolic function matrix comprised of columns of OTUs androws of KEGG Orthology Pathways delineated as described in Example 15.This matrix was restricted to OTUs known to reside in thegastrointestinal tract. From this metabolic function matrix we used thegreedy algorithm described above to design network ecologies capable ofbutyrate production.

Example 23. Identification of Organisms Harboring butyryl-CoA: AcetateCoA Transferase Genes

A panel of putative butyrate forming bacteria can be screened for thepresence of butyryl-CoA: acetate CoA transferase genes to definecandidates for SCFA production. Bacteria are scraped from isolatedcolonies on agar plates or from liquid culture (media selected fromExample 9) and subjected to DNA isolation in 96-well plates. 1 μl ofmicrobial culture or an amount of a bacterial colony approximately 1 μLin volume is added to 9 μl of Lysis Buffer (25 mM NaOH, 0.2 mM EDTA) ineach well of a 96 well, thin walled PCR plate, sealed with an adhesiveseal, and the mixture is incubated at 95° C. for 30 minutes.Subsequently, the samples are cooled to 4° C. and neutralized by theaddition of 10 μl of Neutralization Buffer (40 mM Tris-HCl) and thendiluted 10-fold in Elution Buffer (10 mM Tris-HCl), at which point thegenomic DNA is suitable for use in downstream amplifications such as PCRamplification. Alternatively, genomic DNA is extracted from puremicrobial cultures using commercially available kits such as the Mo BioUltraclean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad,Calif.) or by standard methods known to those skilled in the art.

Degenerate primers are designed to selectively amplify the gene forbutyryl-CoA:acetate CoA transferase based on published genomicsequences. Examples of primers are BCoATforward: 5′GCIGAICATTTCACITGGAAYWSITGGCAYATG (SEQ ID NO: 2047); and BCoATreverse:5′ CCTGCCTTTGCAATRTCIACRAANGC (SEQ ID NO: 2048), where I=inosine; N=anybase; W=A or T; Y=T or C; S=C or G. Amplification is as follows: 1 cycleof 95° C. for 3 min; 40 cycles of 95° C., 53° C., and 72° C. for 30 seach with data acquisition at 72° C.; 1 cycle each of 95° C. and 55° C.for 1 min; and a stepwise increase of the temperature from 55 to 95° C.(at 10 s/0.5° C.) to obtain melting curve data and evaluate productcomplexity. The target amplicon is about 530 nt in length.

Example 24. Identification of Organisms Harboring Butyrate-Kinase Genes

Butyrate may be produced by substrate level phosphorylation ofbutyrylCoA by butyrate-kinase and subsequent phosphorylation of ADP togenerate ATP and butyrate. DNA isolation and PCR amplification wasperformed as in Example 23 with the exception that the following primerswere used: BUKfor: 5′ GTATAGATTACTIR-YIATHAAYCCNGG (SEQ. ID NO: 2049);and BUKrev: 5′ CAAGCTCRTCIACIACIACNGGRTCNAC (SEQ ID NO: 2050), whereI=inosine; N=any base; R=A or G.

Example 25. Identification of Organisms with butyryl-CoA: Acetate CoATransferase Enzymatic Activity

Bacterial strains are grown overnight in an anaerobic chamber at 37° C.in pre-reduced media selected from those described in Example 9. 10 mLof the bacterial culture is harvested by centrifugation at 10,000 rpmfor 10 min, cooled to 4° C. on ice, and disrupted by sonication asdescribed (Duncan, S. et al., 2002 Appl Environ Microbiol Acetateutilization and butyryl coenzyme A (CoA):acetate-CoA transferase inbutyrate producing bacteria from the human large intestine 68: 5186-90).ButyrylCoA: acetate CoA transferase activities are determined by themethod of Barker, scaled for application to a microtiter plate (Barker HA, et al., 1955 Methods Enzymol 1: 599-600).

Example 26. Identification of Organisms with Butyrate-Kinase,Propionate-Kinase and Acetate-Kinase Enzymatic Activity

Bacterial strains are grown overnight in an anaerobic chamber at 37° C.in pre-reduced media selected from those described in Example 9. 10 mLof the bacterial culture is harvested by centrifugation at 10,000 rpmfor 10 min, cooled to 4° C. on ice, and disrupted by sonication asdescribed (Duncan, S. et al., 2002 Appl Environ Microbiol Acetateutilization and butyryl coenzyme A (CoA):acetate-CoA transferase inbutyrate producing bacteria from the human large intestine 68: 5186-90).Butyrate-, propionate-, and acetate-kinase activities were determined bycolorimetric the method of Rose (Rose I A, 1955 Methods Enzymol Acetatekinase of bacteria 1: 591-5).

Example 27. Characterization of Propionate or Butyrate Production from aVariety of Carbon Sources

Strains identified as having either genes for butyrate or propionatefermentation or having the corresponding enzymatic activities areassayed in vitro using a variety of simple carbon sources for theproduction of propionate and butyrate. Bacteria are grown overnight incomplex media selected from Example 9 in an anaerobic chamber at 37° C.When cultures are visibly turbid, the bacteria are pelleted at 10,000×gfor 10 min, the spent media is removed, and they are resuspended inpre-reduced minimal media containing essential vitamins and cofactors(pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, folic acid,nicotinamide, choline, pantothenate, riboflavin or vitamin), divalentmineral salts (including the chloride salts of Mg2+, Ca2+ and Mn2+), andorganic nitrogenous nutrients (especially glycine, glutamate orasparagine) but lacking carbohydrate as a carbon source. Alternatively,strains may be resuspended in a dilution of the original rich media, forinstance a 1:10 or 1:100 dilution, such that essential factors areavailable but a required carbon source is limiting.

Various carbon sources are added to individual cell suspensions. Theseinclude acetate and D and L isomers of lactate, simple sugars includingglucose, galactose, mannose, arabinose, xylose or any other naturallysugar, amino sugars such as N-acetyl glucosamine, galactosamine, sialicacid or glucosamine, sugar alcohols such as glycerol, erythritol,threitol, mannitol, inositol or sorbitol. In addition, the cellsuspensions are individually incubated with complex carbon sourcesincluding di-, tri-, oligo- and polysaccharide carbon sources includingfructans, starches, cellulose, galactomannans, xylans, arabinoxylans,pectins, inulin, and fructooligosaccharides. Tested carbon sources alsoinclude glycopeptides and glycoproteins, such as mucin. The cellsuspension is incubated overnight in a sealed 96-well plate in orderprevent the escape of volatile products.

At the end of the incubation period, the production of propionate,butyrate and other SCFAs is determined according to the followingprotocol:

Reagents

-   -   Internal Standard: 2-ethylbutyric acid, 2-EBA (100 mM)    -   SCFA Mixed Standard: formic acid 10 mM, acetic acid 30 mM,        propionic acid 20 mM, isobutyric acid 5 mM, n-butyric acid 20        mM, n-valeric acid 5 mM, isovaleric acid 5 mM, sodium lactate 10        mM, sodium succinate 10 mM, phenylacetic acid 5 mM    -   MTBSTFA Derivatizing Reagent        (N-Methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide),        purchased from Regis Technologies    -   Concentrated HCl    -   Deionized Water    -   Diethyl ether (unstabilized)    -   Hexane

Linearity Standards Preparation: Linearity Standard 1 is prepared usingstraight SCFA Standard. Linearity Standard 2 is prepared using 100 uL ofSCFA Standard and 900 uL of water. Linearity Standard 3 is preparedusing 100 uL of Linearity Standard 2 and 900 uL of water.

SCFA Extraction:

Extractions of samples (Media and Culture Supernatant), water blanks,and linearity standards were prepared in 4-mL vials using 250 uL ofsample, blank, or standard, 250 uL of concentrated HCl, and 50 uL ofInternal Standard (2-EBA). Once combined, the sample, standards, andblanks were vortexed and allowed to stand for about 5-10 minutes.Diethyl ether (2000 uL) was added to each of the samples, standards andblanks, and each was liquid-liquid extracted for approximately 2minutes. The aqueous and organic phases of the extracted samples,standards and blanks were allowed to separate. Once the layersseparated, 1000 uL of the ether layer was transferred to 2-mLmicro-centrifuge tubes and centrifuged at 14 k for 2 minutes to removeany remaining water.

Sample/Standard/Blank* 250 uL HCl 250 uL Internal Standard (2-EBA) 50 uLEther 2000 uL *substitute deionized water for blank preparations

Derivatization:

Derivatization of all samples, blanks and standards was conducted inHPLC vials using 175 uL of the upper ether layer of samples or standardsand 25 uL of MTBSTFA derivatizing reagent. The reaction mixture wasvortexed and allowed to sit at RT for 24 hours. After 24 hours, theether was removed using a gentle stream of nitrogen, and the residualmaterial was dissolved in 50 uL of hexane. (Note: solvent was removeduntil no further change in volume was apparent, ˜5-10 min). Thederivatized solutions were transferred to small-volume inserts for GC-MSanalysis.

An aliquot of the resulting derivatized material is injected into a gaschromatograph (Hewlett Packard 6890) coupled to a mass spectrometerdetector (Agilent Technologies 5973). Analyses are completed usingDB-5MS (60 m, 0.25 mm i.d., 0.25 mm film coating; P. J. Cobert, St.Louis, Mo.) and electronic impact (70 eV) for ionization. A lineartemperature gradient is used. The initial temperature of 80° C. is heldfor 1 min, then increased to 280° C. (15° C./min) and maintained at 280°C. for 5 min. The source temperature and emission current are 200° C.and 300 mA, respectively. The injector and transfer line temperaturesare 250° C. Quantitation is completed in selected ion monitoringacquisition mode by comparison to labeled internal standards [formatewas also compared to acetate-13C1,d2]. The m/z ratios of monitored ionsfor formic acid, acetic acid, propionic acid, butyric acid, acetate,proprionate and butyrate are as follows: 103 (formic acid), 117 (aceticacid), 131 (propionic acid), 145 (butyric acid), 121 ([2H2]- and[1-13C]acetate), 136 ([2H5]propionate), and 149 ([13C4]butyrate).

At the completion of the experiment, a database is generated for eachtested organism defining what carbon sources yield which SCFAs. In eachcase where a microbe is capable of making propionate or butyrate fromacetate, lactate, a simple sugar including a disaccharide, an aminosugar or sugar alcohol it is scored as positive for SCFA production.Also noted is whether organisms are capable of utilizing complex carbonsources such as polysaccharides to produce SCFA and which SCFAs areproduced.

Example 28. Identification of Organisms Capable of Metabolizing ComplexCarbon Sources Including Polysaccharides and Steroids

Individual strains are screened for their ability to metabolize complexcarbon sources including polysaccharides and steroids (such as bilesalts) according to Example 16 to determine bacterial strain nutrientutilization. For a more complete characterization, specialized platesare constructed utilizing polysaccharide carbon sources includingfructans, starches, cellulose, galactomannans, xylans, arabinoxylans,pectins, inulin, and fructooligosaccharides as well as carbon sourcesincluding glycopeptides and glycoproteins (such as mucin). These can bemade to order by Biolog.

At the end of the experiment, a catalogue of is generated for eachtested organism defining what carbon sources it can utilize.

Example 29. Construction of Cross Feeding Compositions

Data from Examples 27 and 28 are analyzed to determine combinationswhere one organism can make SCFAs from at least one simple carbon sourcebut not from at least one complex carbon source (a polysaccharide or aglycoprotein), and another organism cannot make SCFAs from a simplecarbon source but can utilize at least one complex carbon source as ametabolic substrate.

In these cases, a bacterial mixture is made combining a washed overnightculture of the SCFA producer and a washed overnight culture of the SCFAnon-producer in a minimal media as described in Example 27 with theaddition of the at least one complex carbon source. The bacterialmixture is incubated anaerobically overnight at 37° C. in minimal mediaor a 1:10 or 1:100 dilution of rich media, and the next day is worked upaccording to Example 27 in order to detect whether SCFA has beenproduced. Control cultures include each microbe cultured individually,and the bacterial mixture cultured overnight without the complex carbonsource.

Bacterial mixtures in which control cultures do not yield SCFA but thecomplete mixture does define synergistic bacterial compositions.Synergistic bacterial compositions may be tested for further effects ina variety of in vitro or in vivo models, with and without the complexcarbon source, which may be considered a component of one embodiment ofthe synergistic bacterial composition.

Example 30. In Vivo Validation of Bacterial Composition Efficacy in forAmelioration of Leaky Gut

A murine model for “leaky gut syndrome” is constructed byintraperitoneal injection of pregnant C57BL/6N mice (Charles River,Wilmington, Mass.) with 20 mg/kg poly(I:C) in 200 uL of saline onembryonic day 12.5. Control pregnant mice are injected with 200 uL ofsaline only (Hsiao E Y et al., 2013 Cell Microbiota modulate behavioraland physiological abnormalities associated with neurodevelopmentaldisorders 155: 1451-63).

Pups are randomly selected for treatment with a single bacterialcomposition or a combination of bacterial compositions at the time ofweaning (Day 20-22) and received oral gavage every other day for 6 days.In addition, groups of animals receive mouse chow supplemented with thecomplex carbohydrate relevant to the bacterial composition(s) that is(are) dosed. Control groups (saline injections) receive comparablecombinations of bacterial compositions, with and without the complexcarbohydrate.

Animals are tested at adolescence (3 weeks post-weaning) and adulthood(8 weeks post weaning) for leaky gut. Mice are fasted for 4 hr beforegavage with 0.6 g/kg 4 kDa FITC-dextran (Sigma Aldrich). Four hourslater, serum samples are read for fluorescence intensity at 521 nm usingan xFluor4 spectrometer (Tecan). Increased fluorescence is taken asevidence of leaky gut, while decreased fluorescence is evidence foramelioration of leaky gut induced by poly(I:C) treatment. Preferredbacterial compositions decrease leak gut in mice.

Example 31. In Vivo Validation of Bacterial Composition Efficacy in GermFree Mice Conventionalized with Human Obese Microbiota

Ridaura et al. (2013) showed that germ-free (GF) mice conventionalizedwith microbiota from female twins discordant for obesity showedtaxonomic and phenotypic features of the human donor's microbiota. Micereceiving obese twin microbiota (Ob mice) showed significantly greaterbody mass and adiposity than recipients of lean twin microbiota (Lnmice). Furthermore, they observed that cohousing Ob mice and Ln miceprevented development of the obese phenotype in the Ob mice and showedthat the rescue correlated with invasion of members of the microbiotafrom the Ln mice into the Ob mice.

Ob and Ln mice prepared as described by Ridaura et al. (2013) can beused to test the therapeutic potential of a bacterial composition forobesity. Ob and Ln mice are generated by introducing via oral gavagefecal samples from twins discordant for obesity into 8-9 week old adultmale germ-free C57BL/6J mice. One gnotobiotic isolator is used permicrobiota sample and each recipient mouse is individually caged withinthe isolator. The obese twin must have BMI>30 kg/m2 and the pair musthave a sustained multi-year BMI difference of at least 5.5 kg/m2.Recipient mice are fed a low fat (4% by weight) high in plantpolysaccharide (LF-HPP), autoclaved mouse chow (B&K Universal, EastYorkshire, U.K. diet 7378000).

To prepare the fecal samples for gavage into the GF mice, fecal samplesprovided by donors are frozen immediately after production, stored at−80° C. Samples are homogenized by mortar and pestle while submerged inliquid nitrogen and a 500 mg aliquot of the pulverized material isdiluted in 5 mL of reduced PBS (PBS supplemented with 0.1% Resazurin(w/v), 0.05% L-cysteine-HCl) in an anaerobic Coy chamber (atmosphere,75% N2, 20% CO2, 5% H2) and then vortexed for 5 min at room temperature.The suspension is settled by gravity for 5 min, and then the clarifiedsupernatant transferred to an anaerobic crimped tube that is transportedto a gnotobiotic mouse facility. Prior to transfer of the tube into thegnotobiotic isolator, the outer surface of the tube is sterilized byexposure for 20 min to chlorine dioxide in the transfer sleeve attachedto the isolator. 200 μL aliquot of the suspension is provided into thestomachs of each recipient animal by gavage.

At day 15 post-colonization, the bacterial composition containing atleast 108 CFU/ml per strain is administered daily by oral gavage for 4weeks to half of the Ob mice and half of the Ln mice. The remaining Obmice and Ln mice are administered PBS by the same regimen. Optionally,mice can receive 0-3 days of antibiotic pre-treatment prior toadministration of the bacterial composition. Alternative dosingschedules and routes of administration (e.g. rectal) may be employed,including multiple doses of test article, and 103 to 1010 CFU/ml perstrain of a bacterial composition may be delivered. The bacterialcomposition may optionally be administered together or co-formulatedwith prebiotic(s).

Feces are collected from the cages for analysis of bacterial carriage.Total body weight, fat mass and lean body mass are measured at baselinebefore colonization, at days 8 and 15 post-colonization, and days 22,29, 35, and 42 post-colonization (7, 14, 21, and 28 days afteradministration of the bacterial composition) using quantitative magneticresonance analysis of body composition (EchoMRI-3in1 instrument). Attime of sacrifice, epididymal fat pads are also collected and weighed.Optionally, luminal contents of the stomach, small intestine, cecum, andcolon contents as well as the liver, spleen, and mesenteric lymph nodescan be collected for subsequent analysis. Alternative or additional timepoints may also be collected.

By the end of the treatment period with the bacterial composition, theOb mice receiving the bacterial composition is expected to showsignificant differences in body composition (change in % fat mass; fatpad weight/total body weight) as compared to the Ob group receiving PBSand the Ln groups.

Optionally, at the end of the treatment period, the body composition isdetermined for all mice. Bacterial compositions that produce significantchanges in body composition in the Ob mice (decrease in % fat mass;decrease in fat pad weight; or decrease in total body weight) ascompared to control Ob mice receiving PBS are identified as therapeuticcandidates.

Example 32. In Vivo Validation of Bacterial Composition Efficacy in GermFree Mice Conventionalized With Bacterial Composition and Lean/ObeseMicrobiota Controls

To test the potential of a bacterial composition's ability to treatobesity, 8-9 week old GF C57BL/6J mice can be conventionalized byintroducing by oral gavage a) the bacterial composition, b) fecalsamples from an obese female twin discordant for obesity (obesecontrol), or c) fecal samples from the paired lean female twin (leancontrol). One gnotobiotic isolator is used per microbiota sample andeach recipient mouse is individually caged within the isolator. Theobese twin donors must have BMI>30 kg/m2 and the donating pair must havea sustained multi-year BMI difference of at least 5.5 kg/m2. Recipientmice are fed a low fat (4% by weight) high in plant polysaccharide(LF-HPP), autoclaved mouse chow (B&K Universal, East Yorkshire, U.K.diet 7378000).

To prepare the fecal samples for gavage into the GF mice, fecal samplesprovided by donors are frozen immediately after production, stored at−80° C. Samples are homogenized by mortar and pestle while submerged inliquid nitrogen and a 500 mg aliquot of the pulverized material isdiluted in 5 mL of reduced PBS (PBS supplemented with 0.1% Resazurin(w/v), 0.05% L-cysteine-HCl) in an anaerobic Coy chamber (atmosphere,75% N2, 20% CO2, 5% H2) and then vortexed for 5 min at room temperature.The suspension is settled by gravity for 5 min, and then the clarifiedsupernatant transferred to an anaerobic crimped tube that is transportedto a gnotobiotic mouse facility.

To prepare the bacterial composition for gavage into the GF mice, seeExample 9. Prior to transfer of tubes into the gnotobiotic isolator, theouter surface of the tube is sterilized by exposure for 20 min tochlorine dioxide in the transfer sleeve attached to the isolator. 200 μLaliquots of the fecal suspensions are provided into the stomachs of therecipient animals by gavage.

Feces are collected from the cages for analysis of bacterial carriage.Total body weight, fat mass and lean body mass are measured at baselinebefore colonization, at days 8, 15, 22, 29, and 35. At time ofsacrifice, epididymal fat pads are also collected and weighed.Optionally, luminal contents of the stomach, small intestine, cecum, andcolon contents as well as the liver, spleen, and mesenteric lymph nodescan be collected for subsequent analysis. Alternative or additionaltimepoints may also be collected.

By the end of the treatment period with the bacterial composition, themice receiving the bacterial composition is expected to show bodycomposition (change in % fat mass; fat pad weight/total body weight) andmicrobial composition that is similar to the lean control and that isstatistically different from the obese control.

Example 33. In Vivo Validation of Bacterial Composition Efficacy inDietary Induced Obesity Mouse Model

Male C57BL/6 mice fed a high fat diet can be used to test bacterialcompositions' ability to treat obesity in a diet-induced obesity (DIOmouse) prevention model. To do so, eight groups of mice (n=8) are used,with all combinations of +/− antibiotic pretreatment, bacterialcomposition vs. vehicle, and high fat vs. standard diet.

4 week old male C57BL/6 mice are group housed (2-5 mice per cage) infilter top cages with autoclaved bedding, and free access to autoclavedirradiated food (LabDiet 5053, LabDiet, St. Louis, Mo. 63144) andautoclaved water. For groups receiving antibiotic pretreatment, drinkingwater is replaced by an antibiotic cocktail consisting of 10% glucose,kanamycin (0.5 mg/mL), gentamicin (0.044 mg/mL), colistin (1062.5 U/mL),metronidazole (0.269 mg/mL), ciprofloxacin (0.156 mg/mL), ampicillin(0.1 mg/mL) and vancomycin (0.056 mg/mL) (all constituents fromSigma-Aldrich, St. Louis Mo.) for 1 week, after which autoclaved wateris returned to all cages. The mice are dosed daily with a volume of 0.2ml containing at least 1×108 cfu/ml per strain daily or an equal volumeof sterile PBS. Optionally, the dose may range from 5×106 to 5×1010cfu/ml per strain and or dosing may occur three times a week. After oneweek of dosing, a group (n=10) of mice dosed with vehicle and one withthe bacterial composition are switched to a high fat diet (Research DietD12492) and dosing is continued for all groups. Treatment is continuedfor 15 weeks following the diet shift. Alternative dosing schedules androutes of administration (e.g. rectal) may be employed, includingmultiple doses of test article, and 103 to 1010 CFU/ml per strain of abacterial composition may be delivered. The bacterial composition may beoptionally be administered together or co-formulated with prebiotic(s).

Body weight will be measured three times per week throughout the study.Blood will be drawn by submandibular bleed every three weeks, from whichserum cholesterol and triglycerides will be measured. Fasting bloodglucose will be measured in weeks 12 and 15 following the diet shift. Atsacrifice, total body, gastrocnemius, liver, epididymal fat pad, andcecum weights are measured, and the contents of the cecum as well as onelobe of the liver are stored at −80° C. By the end of the experiment,successful treatments will have statistically significant differences intotal body weight, epididymal fat pad mass, or cholesterol.

Example 34. In Vivo Validation of Bacterial Composition Efficacy inNonobese Diabetic Mouse Model of Type-1-Diabetes

To demonstrate the efficacy of the microbial composition for improvingthe incidence of type 1 diabetes, a type 1 diabetes mouse modeldescribed previously is utilized (e.g. see Markle et al 2013. Sexdifferences in the gut microbiome drive hormone dependent regulation ofautoimmunity. Science 339: 1084). Briefly, nonobese diabetic (NOD)/Jsd(NOD) Specific Pathogen Free (SPF) female mice are housed in sterilizedstatic caging. The animals receive a standard mouse diet (LabDiet #5015,PMI Nutrition International) and autoclaved water. All staff usesautoclaved gowns, caps, masks, shoe covers, and sterile gloves. Animalhandling and cage changes are done under HEPA filtered air. The pathogenstatus is determined by weekly exposure of CD-1 sentinel mice to soiledbedding from the cages in the room. Quarterly serological testing ofsentinels confirmed the NOD mice are negative for: Mouse HepatitisVirus, Minute Virus of Mice, Mouse Parvovirus, Murine Norovirus, SendaiVirus, Theiler's Murine Encephalomyelitis, Retrovirus and for endo- andectoparasites. In addition, live animals are subjected to additional,annual comprehensive testing, including necropsy, histopathology,bacteriology and parasitology testing.

To test the microbial composition for prophylactic ability to reduce,delay or prevent disease appearance, weanling NOD females (aged 22-26days) are gavaged with 250 uL the microbial bacterial composition usinga 24G round tip gavage needle. Recipients are rested for 24 hours, andthis procedure is repeated once. Optionally, mice can receive 0-3 daysof antibiotic pre-treatment prior to administration with the bacterialcomposition. Alternative dosing schedules and routes of administration(e.g. rectal) may be employed, including multiple doses of test article,and 103 to 1010 CFU/ml per strain of a bacterial composition may bedelivered. The bacterial composition may be optionally be administeredtogether or co-formulated with prebiotic(s).

As a negative control, a group of female weanling NOD mice are gavagedwith cecal contents from a female NOD mouse, and as a positive control athird group of female NOD mice are given cecal contents from a male NODdiluted 50× (v/v) and delivered in 250 ul. Spontaneous development ofT1D assessment is assessed biweekly by measuring glucose levels in bloodand urine. Animals are checked daily and are classified as diabetic whenblood glucose exceeds 16 mmol/L or urine glucose exceeds 250 mg/dL.Additionally, serum insulin autoantibody (IAA) is measured by amicro-IAA assay (mIAA). Briefly, 125-I labeled human insulin (PerkinElmer) is incubated with NOD serum with and without cold (unlabeled)human insulin and the immune complex is isolated by binding to protein Aand G Sepharose. The assay is performed on a 96-well filtration plate toretain Sepharose beads and radioactivity is counted on a Topcount96-well plate beta counter or similar instrument. An index is calculatedby taking the difference of cpm between wells without and with coldinsulin. A positive is defined by any conventional cut-off measureincluding a value greater than the 99th percentile of control values, ora value 3 standard deviations beyond the mean of the control values.Furthermore Insulitis is assessed. Briefly, pancreata are dissected andimmediately immersed in OCT media (Tissue-Tek, Torrance, Calif.), frozenin −20° C. 2-methylbutane, and stored at −70° C. Preparation of frozensections is performed with a Leica C M 3050 Cryostat (Leica Canada). Tomaximize analysis of independent islet infiltrates, three 5-μm sectionsare cut at least 400 μm apart. Pancreatic sections are stained withMayer's hematoxylin and eosin Y (H+E, Sigma) to visualize leukocyteinfiltration. Assessment of insulitis severity in pancreatic sections isperformed by one skilled in the art. Briefly, islets are gradedaccording to the following criteria: 0, no visible infiltrates; 1,peri-insulitis as indicated by peri-vascular and peri-islet infiltrates;2, <25% of the islet interior 9 occluded by leukocytes displayinginvasive infiltrates; 3, >25% but <50% of the islet interior invaded byleukocytes; or 4, invasive insulitis involving 50%-100% of the isletfield.

To evaluate the microbial composition for treatment of disease, theprocedure above is repeated whereby NOD nonobese diabetic (NOD)/Jsd(NOD) Specific Pathogen Free (SPF) female mice are housed and evaluatedfor development of diabetes by the criteria described above. Once amouse develops diabetes it is gavaged with the microbial composition,and blood glucose, urine glucose, and insulin serum levels are evaluatedby ELISA weekly to determine disease progression. 7 weeks later animalsare sacrificed and insulitis is evaluated by methods described above.Optionally, mice can receive 0-3 days of antibiotic pre-treatment priorto administration with the bacterial composition. Alternative dosingschedules and routes of administration (e.g. rectal) may be employed,including multiple doses of test article, and 103 to 1010 CFU/ml perstrain of a bacterial composition may be delivered.

Example 35. In Vivo Validation of Bacterial Composition Efficacy in NileRat Model of Type-2-Diabetes

To test the efficacy of a microbial composition for delaying, treatingor preventing the symptoms of type 2 diabetes, a Nile grass rat(Arvicanthis niloticus) model described previously is utilized (e.g. seeNoda, K., et al. (2010). An animal model of spontaneous metabolicsyndrome: Nile grass rat. The FASEB Journal 24, 2443-2453. or Chaabo,F., et al. (2010). Nutritional correlates and dynamics of diabetes inthe Nile rat (Arvicanthis niloticus): a novel model for diet-inducedtype 2 diabetes and the metabolic syndrome. Nutrition & Metabolism 7,29.). Nile rats, which spontaneously develop symptoms of type 2 diabetesand metabolic syndrome, are individually housed and have free access toautoclaved water and autoclaved standard laboratory chow (Lab Diet 5021;PMI Nutrition, St. Louis, Mo., USA). At 5 weeks of age, thrice-weeklydosing of the Nile rats with about 5×108 cfu/ml per strain of themicrobial composition or an equal volume of sterile PBS by oral gavagewhile under light sedation with 50%/50% O2/CO2 is initiated. Optionally,the dose may range from 5×106 to 5×1010 cfu/ml per strain and/or dosingmay occur once weekly. Dosing will continue for 20 weeks postinitiation, optionally lasting 15, 30, 40, or 50 weeks. The model couldbe modified to address prediabetes by shortening the duration to about 3to 10 weeks post initiation of dosing.

Body weight will be measured three times per week throughout the study.Blood glucose, cholesterol, triglycerides, and hemoglobin A1C will bemeasured after obtaining blood by tail bleed while under light sedationwith 50%/50% O2/CO2 every three weeks following initiation of dosing. Atsacrifice, total body, liver, kidney, epididymal fat pad, and cecumweights are measured. Terminal plasma samples are used for measurementof insulin, blood glucose, cholesterol, triglycerides, and hemoglobinA1C. Following perfusion with PBS under deep anesthesia, the liver andkidneys are excised and fixed in 4% paraformaldehyde. Subsequently, 15μm sections are stained with Oil-Red-O and counterstained with Mayer'shematoxylin to facilitate the identification of stores of hydrophobiclipids. The contents of the cecum are flash frozen in liquid nitrogenand stored at −80° C.

Animals treated with successful compositions will have statisticallysignificant differences in terminal body weight, blood glucose,hemoglobin A1C, liver or kidney accumulation of lipid, and/or insulinfrom control animals.

Example 36. In Vivo Validation of Bacterial Composition for ProphylacticUse and Treatment in a Mouse Model of Vancomycin Resistant Enterococcus(VRE) Colonization

The emergence and spread of highly antibiotic-resistant bacteriarepresent a major clinical challenge (Snitkin et al ScienceTranslational Medicine, 2012). In recent years, the numbers ofinfections caused by organisms such as methicillin-resistantStaphylococcus aureus, carbapenem-resistant Enterobacteriaceae,vancomycin-resistant Enterococcus (VRE), and Clostridium difficile haveincreased markedly, and many of these strains are acquiring resistanceto the few remaining active antibiotics. Most infections produced byhighly antibiotic-resistant bacteria are acquired duringhospitalizations, and preventing patient-to-patient transmission ofthese pathogens is one of the major challenges confronting hospitals andclinics. Most highly antibiotic-resistant bacterial strains belong togenera that colonize mucosal surfaces, usually at low densities. Thehighly complex microbiota that normally colonizes mucosal surfacesinhibits expansion of and domination by bacteria such asEnterobacteriaceae and Enterococcaceae. Destruction of the normal floraby antibiotic administration, however, leads to disinhibitionantibiotic-resistant members of these bacterial families, enabling totheir expansion to very high densities (Ubeda et al Journal of ClinicalInvestigation 2010). High-density colonization by these organisms can becalamitous for the susceptible patient, resulting in bacteremia andsepsis (Taur et al, Clinical Infectious Disease, 2012).

To test prophylactic use and treatment of a bacterial composition, a VREinfection mouse model is used as previously described (Ubeda et al,Infectious Immunity 2013, Ubeda et al, Journal of ClinicalInvestigation, 2010). Briefly, experiments are done with 7-week-oldC57BL/6J female mice purchased from Jackson Laboratory, housed withirradiated food, and provided with acidified water. Mice areindividually housed to avoid exchange of microbiota between mice due tocoprophagia. For experimental infections with VRE, mice are treated withampicillin (0.5 g/liter) in their drinking water, which is changed every3 days.

In the treatment model, on day 1, mice are infected by means of oralgavage with 108 CFU of the vancomycin-resistant Enterococcus faeciumstrain purchased from ATCC (ATCC 700221). One day after infection (day1), antibiotic treatment is stopped and VRE levels are determined atdifferent time points by plating serial dilutions of fecal pellets onEnterococcosel agar plates (Difco) with vancomycin (8 ug/ml; Sigma). VREcolonies are identified by appearance and confirmed by Gram staining orother methods previously described (e.g. see Examples 1,2,3, and 4). Inaddition, as previously described (Ubeda et al, Journal of ClinicalInvestigation 2010), PCR of the vanA gene, which confers resistance tovancomycin, confirms the presence of VRE in infected mice. The bacterialcomposition test article such as but not limited to an ethanol treated,gradient purified spore preparation (as described herein), fecalsuspension, or a Network Ecology is delivered in PBS on days 1-3 whilethe negative control contains only PBS and is also delivered on days 1-3by oral gavage. Fresh fecal stool pellets are obtained daily for theduration of the experiment from days −7 to day 10. The samples areimmediately frozen and stored at −80° C. DNA is extracted using standardtechniques and analyzed with 16S or comparable methods (e.g. seeExamples 1 and 2).

In the colonization model, ampicillin is administered as described abovefor day −7 to day 1, treatment with the bacterial composition or vehiclecontrol is administered on day 0-2 and the VRE resistant bacteria at 108CFU are administered on day 14. Fecal samples are taken throughout theexperiment daily from −7 to day 21 and submitted for 16S sequencing aspreviously described (e.g. see Examples 1 and 2).

In both models, titers of VRE in feces are used to evaluate the successof the bacterial composition versus the negative control. A preferredbacterial composition either prevents or reduces colonization by VREcompared to the negative control, or it accelerates the decrease incolonization after cessation of antibiotics. Furthermore, each bacterialcomposition is assessed for the ability of the bacterial compositiontest article to induce a healthy microbiome, as measured by engraftment,augmentation and increase in microbiota diversity.

Example 37. In Vivo Validation of a Bacterial Composition forProphylactic Use and Treatment of a Mouse Model of Carbapenem ResistantKlebsiella (CRKp) Colonization

The emergence of Klebsiella pneumoniae strains with decreasedsusceptibility to carbapenems is a significant threat to hospitalizedpatients. Resistance to carbapenems in these organisms is mostfrequently mediated by K. pneumoniae carbapenemase (KPC), a class Abeta-lactamase that also confers resistance to broad-spectrumcephalosporins and commercially available beta-lactam/beta-lactamaseinhibitor combinations (Queenan et al, Clinical Microbiology Review,2007). KPC-producing K. pneumoniae (KPC-Kp) strains often harborresistance determinants against several other classes of antimicrobials,including aminoglycosides and fluoroquinolones, resulting in trulymultidrug-resistant (MDR) organisms (Hirsch et al, Journal ofAntimicrobial Chemotherapy, 2009). Considering the limited antimicrobialoptions, infections caused by KPC-Kp pose a tremendous therapeuticchallenge and are associated with poor clinical outcomes.

A treatment protocol in a mouse model previously described in micesensitive to KCP-Kp (e.g. Perez et al, Antimicrobial AgentsChemotherapy, 2011) is used to evaluate the bacterial composition (testarticle) for treating carbapenem resistant Klebsiella and reducingcarriage in the GI tract. Female CF1 mice (Harlan Sprague-Dawley,Indianapolis, Ind.) are used and are individually housed and weighedbetween 25 and 30 g. The bacterial composition includes withoutlimitation an ethanol treated, gradient purified spore preparation (asdescribed herein), fecal suspension, or a Network Ecology.

The thoroughly characterized strain of K. pneumoniae, VA-367 (8, 9, 25)is used. This clinical isolate is genetically related to the KPC-Kpstrain circulating in the Eastern United States. Characterization of theresistance mechanisms in K. pneumoniae VA-367 with PCR and DNA sequenceanalysis revealed the presence of blaKPC-3, blaTEM-1, blaSHV-11, andblaSHV-12 as well as qnrB19 and aac(6′)-lb. Additionally, PCR and DNAsequencing revealed disruptions in the coding sequences of the followingouter membrane protein genes: ompK35, ompK36, and ompK37. Antibioticsusceptibility testing (AST) was performed with the agar dilution methodand interpreted according to current recommendations from the Clinicaland Laboratory Standards Institute (CLSI). A modified Hodge test wereperformed, according to a method described previously (e.g. see Andersonet al, Journal of Clinical Microbiology, 2007) with ertapenem,meropenem, and imipenem. Tigecycline and polymyxin E were evaluated byEtest susceptibility assays (AB bioMerieux, Solna, Sweden). Results fortigecycline were interpreted as suggested by the U.S. Food and DrugAdministration (FDA) and according to CLSI recommendations (criteria forPseudomonas) for polymyxin E.

In a prophylactic design, mice (10 per group) are assigned to receiveeither a bacterial composition (test article; e.g. see Example 9 or 10),or control group receiving only the vehicle. After 3 days ofsubcutaneous clindamycin treatment (Day −6, −5, −4) to sensitize them toKPC-Kp, mice are administered the test article or vehicle daily from day−10 to day 0, On day 0, 103 CFU of KPC-Kp VA-367 diluted in 0.5 mlphosphate-buffered saline (PBS) is administered by oral gavage. Fecalsamples are collected 1, 4, 6, and 11 days after the administration ofKPC-Kp to measure the concentration of carbapenem-resistant K.pneumoniae. Fecal samples (100 mg diluted in 800 ml of PBS) are platedonto MacConkey agar with 0.5 ug/ml of imipenem, and the number of CFUper gram of stool is determined. Efficacy of test articles is apparentas a reduction in KPC-Kp burden.

Alternatively other methods may be used to measure the levels ofcarbapenem-resistant K. pneumoniae e.g. PCR, antigen testing, as one whois skilled in the art could perform.

In a treatment design, mice are treated with subcutaneous clindamycin toreduce the normal intestinal flora 1 day before receiving 104 CFU ofKPC-Kp VA-367 by oral gavage. For 7 days after oral gavage with KPC-Kp,mice receive oral gavage of normal saline (control group), or thebacterial composition. Fecal samples are collected at baseline and at 3,6, 8, 11, 16, and 21 days after KPC-Kp VA-367 was given by gavage. Thelevel of CRKp in feces is determined by plating serial dilutions offecal suspensions to MacConkey agar with 0.5 ug/ml of imipenem, and thenumber of CFU per gram of stool is determined. Alternatively othermethods may be used to measure the levels of carbapenem-resistant K.pneumoniae e.g. PCR, antigen testing, as one who's skilled in the artcould perform. Efficacy of test articles is apparent as a reduction inKPC-Kp burden.

Example 38. In Vivo Validation of Bacterial Composition for Efficacy infor the Prophylactic Use or Treatment of Pathogenic Fungus in MiceModels

The bacterial compositions of the invention can be utilized forprophylaxis or treatment of pathogenic fungus in a mouse colonized withone of several Candida species. Adult male CD-1 (ICR) mice areintragastrically inoculated with C. albicans, C. tropicalis or C.parapsilosis as previously described (Mellado et al., DiagnosticMicrobiology and Infectious Disease 2000). Tetracycline-HCl at 1 g/L and5% glucose are included in the drinking water starting on Day −2, 2 daysbefore Candida dosing on Day 0, and throughout the experiment, toenhance colonization. 5×107 Candida are dosed in 0.1 mL on Day 0. By Day4 all mice are colonized as detected by fecal cfu assay described below.Test articles are used in both prophylactic and treatment regimens.Prophylactic dosing with a bacterial composition including withoutlimitation an ethanol treated, gradient purified spore preparation (asdescribed herein), fecal suspension, or a Network Ecology occurs on Day−1 with a dose between 104 and 1010 bacteria, while treatment dosingoccurs on Days 1, 2 and 3 with a similar dose. Negative control groupsin both regimes are dosed with PBS administered in a similar manner. Alltest article dosing is by oral gavage. Treated and untreated mice arekept separate in independently ventilated cages for all of theexperiments. Sterilized food, bedding and bottles are used throughoutthe experiment. Sterilized tap water with or without supplements arealso used to avoid contamination. Starting at day −1 postinfection(p.i.), mice are weighed daily and stool samples are collected from eachanimal and scored for consistency (0, normal feces; 1, mixed stoolsamples containing both solid and pasty feces; 2, pasty feces; 3,semiliquid feces; 4, liquid feces).

Feces are cultured for yeasts. Dilutions of fecal samples are titratedon Sabouraud Dextrose Chloramphenicol agar (Neogen cat #(7306) agarplates which are selective for fungi. After 24-48 h of incubation at 37°C., quantification of the cultures is achieved by counting the platesvisually or by scanning the plates on a Colony Image Analysis Scanner(Spiral Biotech) and processed by the computer software CASBA 4 (SpiralBiotech). The results are noted as colony forming units (CFU) per gramof feces. Effect of bacterial composition on Candida colonization andquality of feces of infected mice is thus analyzed by comparing toplacebo, and representative colonies are submitted for 16S/18S/ITSmicrobial identification before and after infection as previouslydescribed (e.g. See Example 1 and 2).

Using this model, the ability of test articles to prevent fungaldissemination and death is also tested. Starting on Day 4 in the aboveregimen, animals colonized with fungi are treated with immunosuppressiveagents to induce deep neutropenia [defined as >500 polymorphonuclearcell per ml. Total white cells counts are performed using ahemacytometer Neubauer improved (Brand, Werthheim/Main, Germany)]. Theimmunosuppressive agents (150 mg/kg of cyclophosphamide (Sigma) and 65mg/kg of 6-methyl-prednisolone (Sigma) are both administeredintraperitoneally (i.p.) every 72-96 h until deep neutropenia isobtained and continue for 10 days. Test articles are delivered either onDay 4, 5 and 6, in parallel with the start of immunosuppression or for 3consecutive days after deep neurtopenia is confirmed. Control animalsare treated with PBS in each mode of treatment (Day 4-6, or 3 days postneutropenia. Mortality, dissemination and histology are monitored. Whenanimals are severely ill, they are humanely euthanized withpentobarbital (Nembutal) or similar acceptable methods. Dissemination isquantified in kidneys, liver and spleen is quantified by suspendingtissue separately in 2 mL of cooled PBS, and homogenizing using alab-blender (Stomacher 80, Madrid, Spain). Aliquots of the homogenatesare cultured for yeasts and bacterial flora. Results are expressed asCFU per gram of tissues. Candida dissemination is defined as positivecultures of at least two cultured organs. Positive culture is defined asplates yielding a value of >1.5 log 10 CFU/g of tissues. Histologicstudies are also performed on five cut sections of liver, kidneys andspleen to detect yeasts.

Example 39. In Vivo Validation of Bacterial Composition for Efficacy forProphylaxis or Treatment in a Mouse Model of Methicillin ResistantStaphylococcus Aureus (MRSA)

Methicillin resistant Staphylococcus aureus (MRSA) is a Gram positivepathogen that is a major cause of nosocomial infections includingsepsis, pneumonia and surgical site infections. Both nasal andgastrointestinal carriage of MRSA are implicated as sources of organismsassociated with nosocomial infections. Rectal carriage of MRSA is commonin patients in intensive care units and patients with both rectal andnasal colonization had significantly higher rates of MRSA infection thandid patients with nasal colonization alone (Squier et al Staphylococcusaureus rectal carriage and its association with infections in patientsin a surgical intensive care unit and a liver transplant unit. InfectControl Hosp Epidemiol 2002; 23:495-501.)

MRSA is also associated with gastrointestinal disease, includingantibiotic associated diarrhea (Boyce and Havill, Nosocomialantibiotic-associated diarrhea associated with enterotoxin-producingstrains of methicillin-resistant Staphylococcus aureus. Am JGastroenterol. 2005 August; 100(8):1828-34; Lo and Bourchardt,Antibiotic-associated diarrhea due to methicillin-resistantStaphylococcus aureus, Diagnostic Microbiology & Infectious Disease63:388-389, 2009).

A mouse model of MRSA colonization is used to test the efficacy ofbacterial compositions in treating MRSA colonization of the gut. CF1mice are treated with streptomycin (1 mg/ml), delivered in drinkingwater, for 5 days, after which they are orally inoculated with 1e7 cfuMRSA daily from Day 0 to Day 5 via their drinking water (Gries et al,Growth in Cecal Mucus Facilitates Colonization of the Mouse IntestinalTract by Methicillin-Resistant Staphylococcus aureus, JID 2005;192:1621-7). Drinking water is prepared fresh each day. Colonization byMRSA is monitored by determining cfu/ml in feces each day starting onthe day prior to the first day of MRSA inoculation. Feces arehomogenized in sterile PBS and serial dilutions are plated to Mannitolsalt agar and incubated aerobically for 48 h at 37° C. Presumptive MRSAcolonies are confirmed by 16S rDNA PCR and sequencing as in (Examples 1and 2). Bacterial compositions, control PBS or vancomycin are deliveredby oral gavage starting on Day 6 for 3 days. Efficacy is observed as areduction in MRSA cfu/g in feces, and/or faster time to a reduction ofMRSA cfu/g, in the animals treated with bacterial compositions but notin control animals. Efficacy is compared to that of the positive controlvancomycin, which clears the colonization.

The efficacy of bacterial compositions in preventing MRSA colonizationis tested in a mouse model of prophylaxis in which CF1 mice are treatedwith streptomycin, delivered in drinking water, for 5 days. After 2 dayswithout streptomycin, the mice are treated with bacterial compositionsor control PBS by oral gavage for 3 days, and then inoculated with 1e7cfu MRSA by oral gavage. Colonization by MRSA is monitored bydetermining cfu/ml in feces each day starting on the day prior to thefirst day of MRSA inoculation. Feces are homogenized in sterile PBS andserial dilutions are plated to Mannitol salt agar and incubatedaerobically for 48 h at 37° C. Presumptive MRSA colonies are confirmedby 16S rDNA PCR and sequencing as in Examples 1 and 2 for 16Ssequencing. Efficacy is observed as a reduction in MRSA cfu/g in feces,and/or faster reduction of MRSA cfu/g, in the animals treated withbacterial compositions compared to control animals.

Example 40. Clinical Validation of Bacterial Composition for Efficacy inObesity

To demonstrate a bacterial composition's ability to treat obesity, agroup of 400 obese human subjects can be prospectively recruited.Inclusion criteria include BMI 30-45 kg/m². Exclusion criteria includeType 1 or Type 2 diabetes, treatment with any kind of anti-diabetic,anti-hyperglycemic or anti-obesity medication or surgical procedure(e.g. bariatric surgery), significant co-morbidities, participation in aformal weight loss program, either systolic blood pressure >160 mm Hg ordiastolic blood pressure >100 mmHg, subjects whose body weight is notstable, as judged by the Investigator (e.g. >5% change within 3 monthsprior to screening).

During a double blind treatment period of 12 weeks, the experimentaltreatment group (n=200) receives a daily oral dose of about 1×109 CFUsof viable bacteria either in the form of vegetative organisms or sporesor both, whereas the control group (n=200) is administered a placebo atan identical frequency. The composition can be formulated in a delayedrelease enteric coated capsule or co-administered with bicarbonatebuffer to aid passage of viable organisms through the stomach. Thebacterial composition may be optionally be administered together orco-formulated with prebiotic(s).

Patients can be optionally treated with a broad spectrum antibiotic 0-10days prior to first administration of the bacterial composition.Alternative dosing schedules and routes of administration (e.g. rectal)may be employed, including multiple daily doses of test article, and arange of 103 to 1010 CFU of a given composition may be delivered.

At baseline and 6, 12, and 24 weeks after the beginning of the treatmentperiod, change in body weight, waist and hip circumference, andwaist/hip ratio will be measured. By the end of the 24 week treatmentchallenge period, the experimental group is expected to show significantdifferences from the control group in weight loss and/or waist and hipcircumference, optionally 5% or greater weight loss.

Optionally, in the event an effect is detected at the end of the 24 weektreatment period, the durability of the effect may be tested. Allsubjects will be taken off the experimental treatment and change inweight measured after 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 52 weeks.

Example 41. Clinical Validation of Bacterial Composition for Efficacy inWeight Loss

To demonstrate a bacterial composition's ability to cause weight loss, agroup of 400 human subjects with BMI>25 kg/m² is prospectivelyrecruited. Inclusion criteria include BMI>25 kg/m². Exclusion criteriainclude Type 1 or Type 2 diabetes, treatment with any kind ofanti-diabetic, anti-hyperglycemic or anti-obesity medication or surgicalprocedure (e.g. bariatric surgery), significant co-morbidities,participation in a formal weight loss program, either systolic bloodpressure >160 mm Hg or diastolic blood pressure >100 mmHg, subjects whodo not show stable body weight as judged by PI (e.g. >5% change within 3months prior to screening).

During a double blind treatment period of 24 weeks, the experimentaltreatment group (n=200) receives a daily oral dose of about 1×109 CFUsof viable bacteria either in the form of vegetative organisms or sporesor both, whereas the control group (n=200) is administered a placebo atan identical frequency. The composition can be formulated in a delayedrelease enteric coated capsule or co-administered with bicarbonatebuffer to aid passage of viable organisms through the stomach. Thebacterial composition may be optionally be administered together orco-formulated with prebiotic(s).

Patients may be optionally treated with a broad spectrum antibiotic 0-10days prior to first administration of the bacterial composition.Alternative dosing schedules and routes of administration (e.g. rectal)may be employed, including multiple daily doses of test article, and arange of 103 to 1010 CFU of a given composition may be delivered.

At baseline and 6, 12, and 24 weeks after the beginning of the treatmentperiod, change in body weight will be measured. By the end of the 24week treatment challenge period, the experimental group are expected toshow significant differences from the control group in weight loss.

Optionally, in the event an effect is detected at the end of the 24 weektreatment period, the durability of the effect may be tested. Allsubjects will be taken off the experimental treatment and change inweight measured after 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 52 weeks.

Example 42. Clinical Validation of Bacterial Composition for Efficacy inPrediabetes

To demonstrate a bacterial composition's ability to treat prediabetes byexerting beneficial effects on markers associated with the onset ofdiabetes, a group of 60 human subjects with metabolicsyndrome/prediabetes is prospectively recruited. Inclusion criteriainclude either (a) fasting plasma glucose between 5.6 and 6.9 mmol/L and2 hr post-glucose load plasma glucose <7.8 mmol/L, and/or (b) 2 hrpost-glucose load plasma glucose in oral glucose tolerance test (OGTT)between 7.8 and 11.0 mmol/L. Exclusion criteria include establishedgestational, Type 1 or Type 2 diabetes, treatment with any kind ofanti-diabetic, anti-hyperglycemic or anti-obesity medication or surgicalprocedure, use of systemic long-acting corticosteroids or prolonged use(greater than 10 days) of systemic corticosteroids, or any significantmedical condition that would complicate the measurement of the endpointor put the patient at risk.

Optionally, the study can be performed specifically in obese patientsmeeting the above inclusion criteria with the additional inclusioncriteria of BMI 30-45 kg/m² as well as waist circumference >88 cm inwomen and >102 cm in men. Additional exclusion criteria include: 1) ahistory of surgical procedures for weight loss; 2) 2 repeat laboratoryvalues at the screening visit of triglycerides >4.52 mmol; and 3) eithersystolic blood pressure >160 mm Hg or diastolic blood pressure >100mmHg.

During a double blind treatment period of 12 weeks, the experimentaltreatment group (n=30) receives a daily oral dose of about 1×109 CFUs ofviable bacteria either in the form of vegetative organisms or spores orboth, whereas the control group is administered a placebo at anidentical frequency (n=30). The composition can be formulated in adelayed release enteric coated capsule or co-administered withbicarbonate buffer to aid passage of viable organisms through thestomach. The bacterial composition may be optionally be administeredtogether or co-formulated with prebiotic(s).

Patients can be optionally treated with a broad spectrum antibiotic 0-3days prior to first administration of the bacterial composition.Alternative dosing schedules and routes of administration (e.g. rectal)may be employed, including multiple daily doses of test article, and arange of 103 to 1011 CFU of a given composition may be delivered.

At baseline and 4, 8 and 12 weeks after the beginning of the treatmentperiod, glucose tolerance is tested by OGTT and HbA1c (glycosylatedhemoglobin) levels measured. At the same timepoints, insulin secretionwill be assessed by plasma insulin levels measured during the oralglucose tolerance tests. Homeostatic model assessment beta (HOMA-beta)will be used to quantify beta cell function and HOMA-IR for insulinsensitivity. In addition, subjects will perform home blood glucosetesting once weekly at home.

By the end of the 12 week treatment period, the experimental group isexpected to show significant differences from the control group inglucose tolerance, insulin sensitivity, and/or insulin secretionreflecting improved insulin sensitivity, decreased pre-diabetes symptomsand improvement in metabolic syndrome.

Optionally, in the event an effect is detected at the end of the 12 weektreatment period, the durability of the effect may be tested. Allsubjects will be taken off their respective treatment and return fororal glucose tolerance tests after 2 weeks, 4 weeks, 8 weeks, 16 weeks,and 52 weeks to measure HbA1c, insulin secretion, HOMA-beta, andHOMA-IR.

Optionally, the treatment period can be extended to collect anadditional endpoint of progression to type 2 diabetes at 6 months and 12months after the beginning of the treatment period.

Example 43. Clinical Validation of Bacterial Composition for Efficacy inType-2-Diabetes

To demonstrate a bacterial composition's ability to treat type 2diabetes, a group of 60 human subjects with type 2 diabetes isprospectively recruited. Inclusion criteria include diagnosis of type 2diabetes with inadequate glycemic control on diet and exercise,glycosylated hemoglobin between 7.5% and 10.0% at screening, BMI≤45kg/m2.

Exclusion criteria include gestational diabetes, type 1 diabetes,treatment with any kind of anti-diabetic medication in the 12 weeksprior to screening, use of anti-obesity medication/surgical procedure,use of systemic long-acting corticosteroids or prolonged use (greaterthan 10 days) of systemic corticosteroids, or any significantco-morbidities related to the underlying diabetic condition.

Optionally, the study can be done in non insulin dependent type 2diabetics who have inadequate glycemic control who are taking oralmedications such as metformin, sulfonylureas, DPP-4 inhibitors, GLP-1agonists, and SGLT2 inhibitors. Optionally, the study can be done innewly diagnosed non insulin dependent type 2 diabetics who arecompletely treatment naive.

During a double-blinded treatment period of 18 weeks, the experimentaltreatment group (n=30) receives a daily oral dose of about 1×109 CFUs ofviable bacteria either in the form of vegetative organisms or spores orboth, whereas the control group (n=30) is administered a placebo at anidentical frequency. The composition can be formulated in a delayedrelease enteric coated capsule or co-administered with bicarbonatebuffer to aid passage of viable organisms through the stomach. Thebacterial composition may be optionally be administered together orco-formulated with prebiotic(s).

Patients may be optionally treated with a broad spectrum antibiotic 0-10days prior to first administration of the bacterial composition.Alternative dosing schedules and routes of administration (e.g. rectal)may be employed, including multiple daily doses of test article, and arange of 103 to 1010 CFUs of a given composition may be delivered.

At baseline and 6, 12 and 18 weeks after the beginning of the treatmentperiod, HbA1c (glycosylated hemoglobin) levels, fasting plasma glucose,fasting insulin, HOMA-beta, and HOMA-IR, In addition, subjects willperform home blood glucose testing once weekly at home.

Optionally high sensitivity C-reactive protein, adiponectin, totalcholesterol, low-density lipoprotein cholesterol, high-densitylipoprotein cholesterol, triglycerides, systolic and diastolic bloodpressure can also be measured at the same timepoints.

By the end of the 18 week treatment period, the experimental group areexpected to show significant differences from the control group inchange in HbA1c, fasting plasma glucose, insulin sensitivity, and/orinsulin secretion from baseline.

Optionally, in the event an effect is detected at the end of the 18 weektreatment period, the durability of the effect may be tested. Allsubjects will be taken off the experimental treatment and return formeasurement of HbA1c, fasting plasma glucose, fasting insulin,HOMA-beta, and HOMA-IR after 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 52weeks.

Example 44. Clinical Validation of Bacterial Composition for Efficacy inRecent Onset Type-1-Diabetes

To demonstrate a bacterial composition's ability to slow progression ofrecent onset type 1 diabetes, a group of 60 human subjects with recentonset type 1 diabetes is prospectively assembled.

Inclusion criteria include diagnosis of type 1 diabetes within 40 daysprior to screening, positive test for at least one diabetes-relatedautoantibody such as GAD, IA-2, ZnT8, and/or anti-insulin (obtainedwithin 10 days of onset of insulin therapy), peak stimulated C-peptidelevel ≥0.2 pmol/mL following mixed meal tolerance test (MMTT), andevidence of some fraction of residual (normal) pancreatic function.Exclusion criteria include any form of diabetes other than type 1 (e.g.type 2 diabetes), prior or current treatment with corticosteroids,significant co-morbidities.

During a double-blind treatment period of 18 weeks, the experimentaltreatment group (n=30) receives a daily oral dose of about 1×109 CFUs ofviable bacteria either in the form of vegetative organisms or spores orboth, whereas the control group (n=30) is administered a placebo at anidentical frequency. The composition can be formulated in a delayedrelease enteric coated capsule or co-administered with bicarbonatebuffer to aid passage of viable organisms through the stomach. Thebacterial composition may be optionally be administered together orco-formulated with prebiotic(s).

Patients can be optionally treated with a broad spectrum antibiotic 0-10days prior to first administration of the bacterial composition.Alternative dosing schedules and routes of administration (e.g. rectal)may be employed, including multiple daily doses of test article, and arange of 103 to 1010 CFUs of a given composition are delivered.

At baseline and 6, 12 and 18 weeks after the beginning of the treatmentperiod, stimulated C-peptide released in 2 hours during a standard mixedmeal tolerance test (MMTT) and HbA1c levels will be measured. Inaddition, subjects will record total daily dose of insulin in a diary.

By the end of the 18 week treatment period, the experimental group isexpected to show significant differences from the control group inchange in stimulated C-peptide, HbA1c, and/or insulin dosage frombaseline.

Optionally, in the event an effect is detected at the end of the 18 weektreatment period, the durability of the effect may be tested. Allsubjects will be taken off the experimental treatment and return formeasurement of stimulated C-peptide in response to MMTT and HbA1c levelsafter 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 52 weeks.

Example 45. Clinical Validation of Bacterial Composition for Efficacy inReduction of Opportunistic Pathogenic Fungus in Humans

The dimorphic yeast, Candida albicans, is the leading fungal pathogen innormal hosts and in patients with damaged immune systems. Inimmunocompromised hosts such as cancer patients, transplant patients,post-operative surgical patients, premature newborns, or HIV-infectedpeople, C. albicans ranks as the leading fungal pathogen. Invasionleading to systemic infection may also develop in neutropenic patientswhose T cell function is comprised. (Hostetter M K, ClinicalMicrobiology Reviews, January 1994, pp. 29-42.) In this population,disease ranges from aggressive local infections such as periodontitis,oral ulceration, or esophagitis in HIV-infected patients, to complex andpotentially lethal infections of the bloodstream with subsequentdissemination to brain, eye, heart, liver, spleen, kidneys, or bone.Recently, the incidence of systemic candidiasis, which is caused byCandida spp., predominantly Candida albicans, has increased. Thisincrease over the last two decades has caused a rise in the use ofantifungal drugs, including azoles, such as fluconazole or ketoconazol,leading to emergence of resistant organisms and thus increasing the needfor alternative therapies (Looi et al., FEMS Microbiol Lett 2005).

In a prophylactic, randomized, double-blind study, healthy volunteerswho have been prescreened as colonized with Candida albicans at >104cfu/g by fecal culturing are randomized to receive either a placebo or abacterial composition daily. Study volunteers are asked to avoid takingprobiotics in any form in the week prior to dosing. The dosing ofbacterial composition may, optionally, be modified to daily,every-other-day, weekly or any other frequency, and doses may range from105 to 1010 CFU/mL. The subjects provide faecal and vaginal fluidsamples pretreatment and on Days 7, 14 and 28 post-treatment that arecultivated on agar plates within 3 hours after delivery to thelaboratory. Complementary genomic and microbiological methods are usedto characterize the composition of the microbiota from each of thesamples. C. albicans is detected by microbiological methods, for exampleby serial dilution and plating to fungal selective media CHROMagarCandida (BD cat#254093) which selects for fungal organisms, and againstbacterial growth, or another fungal selective media, and also by usingTaqman PCR based assay using similar methods as described previously(Maaroufi et al., J Clin Microbiol. 2003). A reduction in C. albicanslevels in feces indicates efficacy in reducing colonization.

SUMMARY

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to beobtained. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

The foregoing description of the embodiments of the invention has beenpresented for the purpose of illustration; it is not intended to beexhaustive or to limit the invention to the precise forms disclosed.Persons skilled in the relevant art can appreciate that manymodifications and variations are possible in light of the abovedisclosure.

Some portions of this description describe the embodiments of theinvention in terms of algorithms and symbolic representations ofoperations on information. These algorithmic descriptions andrepresentations are commonly used by those skilled in the dataprocessing arts to convey the substance of their work effectively toothers skilled in the art. These operations, while describedfunctionally, computationally, or logically, are understood to beimplemented by computer programs or equivalent electrical circuits,microcode, or the like. Furthermore, it has also proven convenient attimes, to refer to these arrangements of operations as modules, withoutloss of generality. The described operations and their associatedmodules may be embodied in software, firmware, hardware, or anycombinations thereof.

Any of the steps, operations, or processes described herein may beperformed or implemented with one or more hardware or software modules,alone or in combination with other devices. In one embodiment, asoftware module is implemented with a computer program productcomprising a computer-readable medium containing computer program code,which can be executed by a computer processor for performing any or allof the steps, operations, or processes described.

Embodiments of the invention may also relate to an apparatus forperforming the operations herein. This apparatus may be speciallyconstructed for the required purposes, and/or it may comprise ageneral-purpose computing device selectively activated or reconfiguredby a computer program stored in the computer. Such a computer programmay be stored in a tangible computer readable storage medium or any typeof media suitable for storing electronic instructions, and coupled to acomputer system bus. Furthermore, any computing systems referred to inthe specification may include a single processor or may be architecturesemploying multiple processor designs for increased computing capability.

Embodiments of the invention may also relate to a computer data signalembodied in a carrier wave, where the computer data signal includes anyembodiment of a computer program product or other data combinationdescribed herein. The computer data signal is a product that ispresented in a tangible medium or carrier wave and modulated orotherwise encoded in the carrier wave, which is tangible, andtransmitted according to any suitable transmission method.

Finally, the language used in the specification has been principallyselected for readability and instructional purposes, and it may not havebeen selected to delineate or circumscribe the inventive subject matter.It is therefore intended that the scope of the invention be limited notby this detailed description, but rather by any claims that issue on anapplication based hereon. Accordingly, the disclosure of the embodimentsof the invention is intended to be illustrative, but not limiting, ofthe scope of the invention, which is set forth in the following claims.

Tables

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LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US10881696B2).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method of treating or reducing the severity ofa disorder selected from the group consisting of Clostridium difficileinfection and ulcerative colitis, comprising: administering to amammalian subject in need thereof an effective amount of a therapeuticbacterial composition, the therapeutic bacterial composition comprisinga plurality of isolated bacteria, wherein the plurality of isolatedbacteria comprises: a first bacterial operational taxonomic units (OTU)comprising a 16S rDNA comprising a sequence at least 97% identical toSEQ ID NO: 774, a second bacterial OTU comprising a 16S rDNA comprisinga sequence at least 97% identical to SEQ ID NO: 856, a third bacterialOTU comprising a 16S rDNA comprising a sequence at least 97% identicalto SEQ ID NO: 880, and a fourth bacterial OTU comprising a 16S rDNAcomprising a sequence at least 97% identical to SEQ ID NO:
 1670. 2. Themethod of claim 1, wherein the first bacterial OTU comprises a 16S rDNAcomprising SEQ ID NO: 774, the second bacterial OTU comprises a 16S rDNAcomprising SEQ ID NO: 856, the third bacterial OTU comprises a 16S rDNAcomprising SEQ ID NO: 880, and the fourth bacterial OTU comprises a 16SrDNA comprising SEQ ID NO:
 1670. 3. The method of claim 1, wherein thetherapeutic bacterial composition is substantially depleted of aresidual habitat product of a fecal material.
 4. The method of claim 1,wherein the therapeutic bacterial composition is capable of inducing theformation of IgA, RegIII-gamma, IL-10, regulatory T cells, TGF-beta,alpha-defensin, or beta-defensin in the mammalian subject.
 5. The methodof claim 1, wherein the therapeutic bacterial composition is provided asan oral finished pharmaceutical dosage form including at least onepharmaceutically acceptable carrier.
 6. The method of claim 5, whereinthe oral finished pharmaceutical dosage form comprises at least about1×10⁴ colony forming units of each of the first, second, third, andfourth bacterial OTUs per dose of the composition.
 7. The method ofclaim 5, wherein the oral finished pharmaceutical dosage form is acapsule.
 8. The method of claim 5, wherein the oral finishedpharmaceutical dosage form is a tablet.
 9. The method of claim 5,wherein the oral finished pharmaceutical dosage form is a powder. 10.The method of claim 1, wherein the therapeutic bacterial composition isprepared by ethanol treatment.
 11. The method of claim 1, wherein thetherapeutic bacterial composition is prepared by heat treatment.
 12. Themethod of claim 1, wherein the therapeutic bacterial composition isprovided as an oral finished pharmaceutical dosage form comprising atleast 1×10⁴ colony forming units of each of the first, second, third,and fourth bacterial OTUs per dose of the composition and including atleast one pharmaceutically acceptable carrier and the therapeuticbacterial composition is prepared by ethanol treatment.
 13. The methodof claim 1, wherein the therapeutic bacterial composition is provided asan oral finished pharmaceutical dosage form comprising at least 1×10⁴colony forming units of each of the first, second, third, and fourthbacterial OTUs per dose of the composition and including at least onepharmaceutically acceptable carrier and the therapeutic bacterialcomposition is prepared by heat treatment.
 14. The method of claim 1,wherein the therapeutic bacterial composition is provided as an oralfinished pharmaceutical dosage form comprising at least 1×10⁴ colonyforming units of each of the first, second, third, and fourth bacterialOTUs per dose of the composition and including at least onepharmaceutically acceptable carrier and the therapeutic bacterialcomposition is prepared by ethanol treatment, and wherein thetherapeutic bacterial composition is substantially depleted of aresidual habitat product of a fecal material.
 15. The method of claim 1,wherein the therapeutic bacterial composition is provided as an oralfinished pharmaceutical dosage form comprising at least 1×10⁴ colonyforming units of each of the first, second, third, and fourth bacterialOTUs per dose of the composition and including at least onepharmaceutically acceptable carrier and the therapeutic bacterialcomposition is prepared by heat treatment, and wherein the therapeuticbacterial composition is substantially depleted of a residual habitatproduct of a fecal material.
 16. The method of claim 1, wherein thetherapeutic bacterial composition is provided as an oral finishedpharmaceutical dosage form comprising at least 1×10⁴ colony formingunits of each of the first, second, third, and fourth bacterial OTUs perdose of the composition and including at least one pharmaceuticallyacceptable carrier and wherein the oral finished pharmaceutical dosageform is a capsule, a tablet, or a powder.
 17. The method of claim 1,comprising administering an antibiotic to the mammalian subject prior toadministering the therapeutic bacterial composition.